Bacteriology - General and Diagnostics Flashcards
Sterilisation - Chemical
Gas - Ethylene dioxide
Plasma - H2O2
Liquid - Glutaraldehyde, Iodine, Potassium-Iodide, Formaldehyde, Peracetic acid.
Comparison of Prokaryotes and Eukaryotes :
Cell wall
Prokaryotes: Usually present chemically complex (Peptidoglycan)
Eukaryotes: When present chemically simple (Plants)
Exotoxins - Inhibition of Neurotransmitter release, 2 Examples
1) Tetanospasmin - SNARE Proteins disruption on Renshaw and α Motor Synapse (GABA): Muscle Rigidity
2) Botulinum Toxin - SNARE Proteins disruption on NMJ (ACh) : Muscle Flaccid
Bacterium Strain
population of bacteria originated from one colony maintained for a long time during laboratory condition with constant genetic properties.
Principles of Antibacterial Chemotherapy -
Dysbacteriosis, and Prevention.
Dysbacteriosis - microbial imbalance or maladaptation on or inside the body, such as an impaired microbiota. For Prevention - Use as Narrow spectrum Antibiotics as Possible!
What are the 3 major steps of entry to host organism?
Adhesion, Penetration and Invasion
Immunosuppression is usually caused by ____ or _____ but can happen in the case of ________ or leprosy as well.
Immunosuppression is usually caused by Viruses or Protozoa but can happen in the case of Tuberculosis or leprosy as well.
Chemical and Biological Methods of Control for Sterilization
Chemical - Indicative Dyes
Biological - Bacillus Stearothermophilus Spores
Principles of Antibacterial Chemotherapy -
Duration of Treatment
Minimum - 3 days, In case of No recovery:
Acute Infection - 5 days<
Severe Infection - 8-14 Days
Sepsis/Endocarditis - 3-6 Wks
Definition of Pathogen
Microorganism that is able to cause disease in a plant, animal or insect.
Methods of Determination of Bacteria
Culturing
Direct - Macroscopic Smears, Antigen Detection, Enzyme or Nucleic acid oriented
Indirect - Detection of Pathogen by specific Antibodies
Neisser Staining: Microscopic Image
Rods with Club ends (Purple) and Yellow Body
חצופים בקלאב הוטל ה”סוף הסגול “ העיקר גוף צהוב
History of Microbiology - Sir Alexander Fleming
1928
Discoverer of Lysozyme and Antibiotic Substance Penicillin G
Facultative/ non-obligatory structures that some bacteria have:
Capsule
Flagela
Pilli
Spore
Heterotrophic Bacteria
Use organic compounds for growth
logic of use of Ampicillin and Gentamicin together:
Ampicillin for cell wall
Gentamicin for ribosomal inhibition (aminoglycoside)
Comparison of Prokaryotes and Eukaryotes:
Chromosome
Prokaryotes: usually circular chromosome, Histones-like proteins, Plasmids, No introns in mRNA
Eukaryotes: Multiple linear chromosomes with Histones and all genes contain Introns
_______ Antibody have low ______ with the pathogen and do not produce a strong enough immune response, they might even block _____ antibodies from binding the pathogen.
Ineffective Antibody have low Avidity with the pathogen and do not produce a strong enough immune response, they might even block effective antibodies from binding the pathogen.
Exotoxins - Lysis of Cell Membrane, 2 Examples
1) C. Perfringens Alpha Toxin - PLC causing Myonecrosis
2) S. Pyogenes Streptolysin - RBCs Beta Hemolysis
Resistance Abbreviations:
PRP
Resistance Abbreviations:
PRP = Penicillin Resistant Pneumococcus
Classification of Exotoxins based on toxicity mechanism.
Extracellularly-acting toxins - 2 Examples
- Membrane damage: pore formation
- Superantigens: TSST
Where is it most probable ro find LTA?
Gram Positive Bacteria cell wall
Symptomless Infections - Possible Causes
Try to remember at least 3
1) Fast Elimination or Lifelong Immunity
2) Latent Infection - Activation of Pathogen is delayed
3) Persistent Infection - Viral Form of Latency: Episomal (Herpes) or Integrated (Retrovirus)
4) Carrier State
What are the 4 major shapes of bacteria?
Cocci - spherical
Bacilli - rods
Spiral
Pleomorphic
What is the essential difference between Obligate and Facultative pathogen Microbes? regarding treatment and transmission
Obligate Pathogen - Never part of the Normal body flora. must be eradicated.
Facultative - usually invade unnatural areas for it within opportunities (part of the normal flora).
Order of Taxonomy:
Kingdom → _______ → Class → ______ → Family → Genus → ______
Order of Taxonomy:
Kingdom → Phylum → Class → Order → Family → Genus → Species
Immunochromatography Samples possible
Serum, Whole Blood, Plasma, CSF, Stool, Spit
Size range of Bacteria
Specific sizes of Cocci and Bacilli
300 nanometer to 20 micrometer
Cocci are around 1 micron size
Bacilli could be up to 10 microns
(Spirochetes can be longer)
Heat Disinfection:
Tyndallization
consists of heating to 85oC for 30 minutes
with subsequent incubation at 37oC in repeated cycles several times.
What is the advantage in using Exotoxins in vaccines in contrast to Endotoxins?
Exotoxins can be inactivated to become Toxoids
Resistance of microbes: largest and smallest
Prions, Bacterial Spores and Mycobacteria are the most resistant.
Gram positive bacteria and viruses are the least
Antibiotic therapy: 2 kinds
1) Empirical - based on available data, no time.
2) Direct/Rational - lab specific antibiotic, investigation of causative agent before prescription
Mechanism of evasion - Avoiding contact with Phagocytes: VFs that
_______ phagocyte chemotaxis even in ____ ___ ____ ____ ___________ .
Mechanism of evasion - Avoiding contact with Phagocytes: VFs that
Inhibit phagocyte chemotaxis even in very low concentration .
Administration of Neisseria Meningitidis polysaccharide capsule vaccine it is conjugated to the diphtheria toxoid to elevate the effectiveness of the vaccine. This is due to the fact that the toxoid contains key _____ ____ Components .
Administration of Neisseria Meningitidis polysaccharide capsule vaccine it is conjugated to the diphtheria toxoid to elevate the effectiveness of the vaccine. This is due to the fact that the toxoid contains key Amino Acids Components .
History of Microbiology - Louis Pasteur
1870
Discovered the principles of vaccination + Vaccinations for Rabies, Anthrax and Chicken Cholera
Discovered Microbial fermentation and Pasteurization.
Determination of MIC - Disc diffusion test
1)Preparation of lawn culture (Flooded Equal Bacterial Growth Solid Media)
2)Placing paper discs covered with antimicrobial drug
3)37°C overnight
4)Measuring the diameter of the zone of inhibition
around the discs
Bacteria that are not lysed and killed in serum by MAC are called ____ ____ .
Bacteria that are not lysed and killed in serum by MAC are called Serum Resistant.
________ is a polypeptide antibiotic that causes alteration in ion permeability of gram negative bacteria.
Polymyxin is a polypeptide antibiotic that causes alteration in ion permeability of gram negative bacteria.
Capsule Stain: Preparation process and Purpose
לקחתי קפסולה אחי בהודו משהו שלילי אחי משהו דארק סלייד אחי. תשמע התחממתי אז נוסע לפושקין דקה לשים קצת מים ושמן, השקעה אחי.
- Drop of India Ink spread with other slide
- Heat fix
- Fuchsine for 1 minute
- Water rinse and Oil Immersion for observation
Plasma Sterilization (forms Hydrogen Peroxide radicals): Protocol:___C in ___ for ___ .
Plasma Sterilization (Hydrogen Peroxide radicals): Protocol:30-60C in Vacuum for Hour .
PFGE - Pulse Field Gel Electrophoresis
Epidemiological - Bacterial DNA detected and divided by Size (specific cleavage sites)
Which Bacteria type are resistant to Lysozymes?
Gram Negative
Materials for Gram staining
Crystal Violet with sodium oxalate
Potassium-Iodide (Lugol Solution)
Ethanol
Safranin
Mechanism of evasion - Avoiding contact with Phagocytes:
_______ the antigenic surface of the bacterial cell by __________ with a ______ like component .
Mechanism of evasion - Avoiding contact with Phagocytes:
Hide the antigenic surface of the bacterial cell by covering with a self like component .
Penicillin - examples for bacteria effective against
T. Paliidum, S. Pneumonia
Generation time
The time between two Bacterial Binary Fission
Chemotherapeutic Index - DTM/DCM
Ehrlich’s
“Dosis Tolerate Maxima/Dosis Curative Minima”
Maximal Host Tolerable Dose Divided by Minimal Pathogen Killing Dose
Hanging Drops: Preparation process and Purpose
- drop of bacterial suspension in the middle of slide
- Vaseline on the Rim of hollow slide cavity (border)
- Cover and Invert then fast reverse
- low power magnification shows Motility of bacteria
Alcohols used for Disinfection:
_____ (80%), ____ (60%), _____ (70%)
Alcohols used for Disinfection:
Ethanol (80%), Propanol (60%), Isopropanol (70%)
Monotrichous, Polytrichous, Lofotrichous, Amfitrichous, Lofoamfitrichous are all examples for? What Antigen do they represent?
Types of Flagella
H antigen
Generations of Cephalosporins - gradual changes
From 1st to 5th - less GRAM+ more GRAM -
Precipitation
Ag-Ab bound together barely visual
Sterilisation - Steam (Moist Heat / Autoclaving) :
+1 atm 121c 30 min
or
+2 atm 134c 10 min
When there is a patient with a Deep wound contaminated with soil he will receive Passive Immunization with IgG through injection to prevent the risk of___.
When there is a patient a deep wound contaminated with soil he will receive Passive Immunization with IgG through injection to prevent the risk of Tetanus.
Portal of Entry:
The ___ Normally serves as a barrier for Infections, However any ___, ___ or Invasive Procedures invite ___ Entrance.
Portal of Entry:
The Skin Normally serves as a barrier for Infections, However any Wounds, Catheters or Invasive Procedures invite Pathogen Entrance.
Examples for naming the grouping kinds of the bacteria?
Diplococci - pairs of Cocci
Staphylococci - grape like clusters of cocci
Streptococci - chains or cocci
Obligate Aerobe Bacteria have _____ , _____ and _____ ______ that allow them to use Oxygen as the final electron acceptor in the ETC without suffering from ROS.
Obligate Aerobe Bacteria have Catalase , Peroxidase and Superoxide Dismutase that allow them to use Oxygen as the final electron acceptor in the ETC without suffering from ROS.
Conjugated vaccine:
- BCG vaccine
- DPT vaccine
- Haemophilus influenzae vaccine
- Epidemic typhus vaccine
- S. pneumoniae 23 valent vaccine
Haemophilus influenzae vaccine
History of Microbiology - Antoine van leeuwenhoek
1676
“Father of Microbiology”
Improved the microscope for cell level inspection
Key components of Gram (-) bacteria cell wall
LPS - Lipopolysaccharide (Lipid A and O antigen)
Periplasmic space with beta Lactamase
(Outer membrane present and much less peptidoglycan)
What are important intracellular enzymatic reactions products in bacteria? Importance?
Indole, Ammonium, H2S
Detection by indicators and diagnosis
Antigenic ______ on ______ allows for obsoletion of the immune response. No Antibody-Antigen Interaction occurs after first encounter.
Antigenic variation on fimbriae allows for obsoletion of the immune response. No Antibody-Antigen Interaction occurs after first encounter.
Bacteria that can enter ___ ___ or Penetrate ____ ___ and spread from Initial site of entry are Invasive.
Bacteria that can enter Host cells or Penetrate Mucosal Mem. and spread from Initial site of entry are Invasive.
How can we study of the resistance of microbes to antimicrobial agents?
Determination of MIC: E –test, Microdilution test, Agar plate dilution test….
Infection Transmission routes - Horizontal Spread
Iatrogenic Spread
Infection transmission during Medical examination/Procedure (e.g. Klebsiella Pneumoniae , S. Epidermidis..)
Superantigens cause activation of ___% of _ ___ which causes elevation in __, __ and __ which can ultimately cause ___ ___.
Superantigens cause activation of 2-20% of T cells which causes elevation in TNFα, IFNγ and IL 2 which can ultimately cause Septic Shock.
4 Entry portal for Pathogens:
1) Respiratory Tract
2) Gastrointestinal Tract
3) Urogenital Tract
4) Skin - Cut/Punctured or Injured
Exotoxins - Inhibition of Phagocytosis, Example
Pertussis Toxin - cAMP↑ in Neutrophills causing Impaired recruitment and Lymphocytosis
Obligate intracellular parasites - Culturing:
Treponema pallidum spp. Pallidum
Can grow in rabbit testis
Pasteur Effect
Upon giving oxygen to Facultative Anaerobes Bacteria the fermentation rate decreases and Oxidative Phosphorylation Increases.
Obligatory Vaccines (7 in Total) : ____, ____, IPV, ____, _____, MMR, HepB.
Obligatory Vaccines (7 in Total) : BCG, DPTa, IPV, HiB, PCV13, MMR, HepB.
Mechanism of evasion - Survival inside phagocytes:
_____ from Phagosome is crucial for some intracellular pathogens.
Mechanism of evasion - Survival inside phagocytes:
Escaping from Phagosome is crucial for some intracellular pathogens.
______ _____ happens when a pathogen coats itself with host ______ or ________ to hide their own antigen surfaces.
Antigenic Disguise happens when a pathogen coats itself with host proteins or polysaccharides to hide their own antigen surfaces.
________ binds ______ in membranes and causes Lysosomal granules explosion in cell .
Streptolysin O binds cholesterol in membranes and causes Lysosomal granules explosion in cell .
What are the advantages of biofilm type of culturing?
Flexibility
Nutrient trap
O2 take up
Protection from phagocytosis or antibiotics
______ is a ______ vaccine for Tuberculosis.
BCG is an attenuated vaccine for Tuberculosis.
Aminopenicillines
Spectrum and examples
Extended Spectrum - GRAM Positive and GRAM Negative
Ampicillin and Amoxicillin
Horizontal Gene Transfer: Lytic and Lysogenic phases are leading to _________ of genetic Material.
Horizontal Gene Transfer: Lytic and Lysogenic phases are leading to Transduction of genetic Material.
How long does spore germination takes?
90 minutes~
Comparison of Prokaryotes and Eukaryotes:
Sexual Recombination
Prokaryotes: None, transfer of DNA only
Eukaryotes: Meiosis
How do Cationic Detergents work?
interfere with cell membrane permeability, causing leakage of nitrogen and phosphorus containing compounds from the cell.
Ethylene oxide gas - Action and Use
Alkylating agent. Disinfectant used to sterilize heat-sensitive objects such as rubber and plastics.
What is this Vaccine for? and When is it recommended?:
BCG
(Extra knowledge)
BCG - TB
0-6 weeks (in Hospital usually )
Mechanisms of Bacteria Evading Complement:
Components of Capsules - 2 Examples
Polysaccharide Capsules
Sialic Acid
What are the 3 important constituents of pharmacokinetics?
- Serum concentration
- Half time
- side effects
Infection Transmission routes - Horizontal Spread
Vehicles for Transmission, 3 Examples
1) Air-borne: Coughing, Sneezing, Talking (Droplets)
2) Contaminated Water or Food
3) Blood Sucking Insects
Obligate intracellular parasites - Culturing:
Rickettsiae
In embryonated egg or tissue culture, the risk of laboratory acquired infection is extremely high, so culture rarely done
Superantigens bind to ___ _____ ___ molecules at a location in the outside the groove, upon antigen presentation they then bind to a part of the ______ __ ____ that is outside the normal antigen-binding site this trigger T-cell activation on massive scales.
Superantigens bind to MHC Class II molecules at a location in the outside the groove, upon antigen presentation they then bind to a part of the β-chain of TCR that is outside the normal antigen-binding site this trigger T-cell activation on massive scales.
LPS - Hemostatic Effect
Induction of Tissue Factors in Monocytes - Coagulation↑ and DIC Risk↑
On which bacterial strains we will be able to find Exotoxins production?
Multiple strains on Gram Positive and Gram Negative Spectrum
Subunit Vaccines (of Viruses): 3 Examples
Subunit Vaccines (of Viruses):
1) HepB Surface Proteins
2) HPV Capsid Proteins
3) Influenza hemagglutinin and neuraminidase
Bacitracin, Cycloserine and Fosfomycin are all -
Cell Synthesis inhibitor Antibiotics
3 Components of LPS
1) Polysaccharide chain
2) Lipid A
3) O antigen
_______ is a lipopeptide antibiotic that alters the permeability of gram positive bacteria and leads to leakage of cytosolic contents.
Daptomycin is a lipopeptide antibiotic that alters the permeability of gram positive bacteria and leads to leakage of cytosolic contents.
Essential structures for bacteria
Cell wall
Cell membrane
Cytoplasm
Nuclear material
What are the different Temperature Demands of Bacteria? (3 categories to remember)
Psychro- / Meso- / Thermo- philic
15-20C / 30-37C / 50-60C
Antibiotics that act against synthesis of Nucleic acids
Sulfonamides
Trimethoprim with sulfamethoxazole
Complement Fixation test
Indirect Method - presence of specific Ab is checked
Ag-Ab complex causes Complement aggregation preventing lysis of the Sheep RBCs by MAC :
Lysed RBCs - NEGATIVE
No Lysis, RBCs Intact - POSITIVE
Primary vs. Secondary Infections
Primary Infections - develop in healthy individuals
Secondary Infection - Develop in Individuals already infected with another pathogen.
How long does spore formation takes?
6-8 hours
Color differences in cultures could be explained by ?
Hydrophilic pigment - diffuses to media
Hydrophobic doesn’t
Comparison of Prokaryotes and Eukaryotes :
Cell Size
Prokaryotes:0.2 - 2 Microns
Eukaryotes:7-100 Microns
Why should we use only 50-70 % ethanol (not 100) as a detrgent ?
100% causes quick denaturation of bacterial wall protein forms a protective shell
Homologous recombination
The incorporation of the transformed donor DNA into the host bacteria DNA.
Key point of view establishing new antibiotics resistance.
Pilus function
Attachment or sexual - for genetic material exchange
Examples for Industrial Use of Microbiology in Fermentation
Yeast - Beer, Wine, Bread
Bacteria - Yogurt, Cheese, Sour Cream
What are spores?
Lack of nutrients trigger its formation - dormant form.
Survivng harsh conditions: including heat aciditiy or lack of oxygen
What is the medical importance of the difference in ribosomal subunits between pro and eukaryotes?
Antibiotics could be applied to attack only the infecting bacteria e.g. Tetracycline attacking bacterial 30s subunit.
What are the categories for classifying bacterias in the nature (by order from larger to smaller)?
Domain-Kingdom - Phylum - Class - Order - Family - Genus - Species
“Dumb Kids Prefer Cheese Over Fresh Green Spinach “
Exotoxins - Increasing Fluid Excretion, 3 Examples
1) ETEC - Heat Labile + Stabile toxins - cAMP↑ + cGMP↑
2) B. Anthracis - Edema Toxin - cAMP↑
3) V. Cholera - Cholera Toxin - cAMP↑
How does Teichoic acid aids Gram positive bacteria ?
Resistance for Lyzozymes
Horizontal gene transfer
Transmission of genetic material by plasmids in the sex pills conduction between different bacteria. Could help form new immunity to prescribed antibiotics (importance of intake schedule).
Enzymes produced by obligate aerobes:
- Oxidase, catalase, peroxidase
- Catalase, superoxide-dismutase, urease
- Superoxide-dismutase, peroxidase, catalase
- Peroxidase, catalase, oxidase
- Hydrogenase, oxidase, catalase
Superoxide-dismutase, peroxidase, catalase
Steps in preparation and Fixation of bacterial smears:
General Process - 3 steps
1 - Let dry on slide in room temp
2 - Identify slide on the backside with marker
3 - Fix by 3 times with Bunsen burner or methanol
What are bottle cultures used for ? (Which samples)
Name one advantage of these culture types?
Blood and CSF
CO2 detection signaled
Koch’s Postulate - Criteria 1:
The Microorganism must be ___ __ ____ in all organisms ___ __ ____ but not in____ .
Koch’s Postulate - Criteria 1:
The Microorganism must be found in abundance in all organisms suffering from the disease but not in healthy ones.
Koch’s Postulate - Criteria 2:
The Microorganism must be ___ from the diseased organism and _______ .
Koch’s Postulate - Criteria 2:
The Microorganism must be Isolated from the diseased organism and grown in pure culture.
Antiseptics:
Ethanol (50-70%) - Action and Use
Denaturation of Proteins and Solubilization of Lipids.
On Skin.
Mechanisms of Bacteria Evading Complement:
Pseudomona aeruginosa causes destruction of components of complement system with _______ _____ .
Mechanisms of Bacteria Evading Complement:
Pseudomona aeruginosa causes destruction of components of complement system with Extracellular Elastase .
Examples for Use of Animals in the Lab for Microbial Investigation:
-“Harmless probe”- biological activity test
LAL (blood cells of Limulus polyphemus) test is based on the gel clot method- when the Limulus amebocyte lysate (LAL) Shows Presence of bacterial endotoxin lipopolysaccharide (LPS) in sample.
MALDI-TOF
Matrix assisted laser desorption/Ionization, Time of Flight
Culturing and using the machine to identify by Ionization of the bacterial lysed biochemical components.
Very efficient.
____ forms of bacterial antigens are able to “______” antibodies before they arrive to the bacterial cells.
Soluble forms of bacterial antigens are able to “neutralize” antibodies before they arrive to the bacterial cells.
Kinds of Hemolysis?
Alpha - incomplete green halo on blood or choclate agar.
Beta - complete transparent halo on blood agar
Gamma - no hemolysis
What amino acid depletion contributes the most for the initiation of the cascade that allows for spore formation?
Alanine
Motility test Medium, how does it enable detection?
Semisolid Material suspension with straight line stab - Turbidity after incubation means bacteria moved away and growed.
Disseminated Infection has extended beyond its origin and involved the ____ or ___ systems to “seed” other areas in the body.
Disseminated Infection has extended beyond its origin and involved the Bloodstream or Lymphatic system to “seed” other areas in the body.
ID50/LD50/TCID50 Measurements Gave a value around 1 -10^2 Germs.
What does it mean?
High Virulence since this is a Low Germ count that causes 50% of Hosts/Cultures to have a pathologic conditions (Infection/Death/Damage).
LD50
A Quantitative Determinant of Virulence
Lethal Dose - Number of Germs causing death in 50% of Hosts.
What are the kinds if bacterial growth?
Planktonic - liquids
Colony - Solid
Biofilm - Surface (poor nutrition)
Comparison of Prokaryotes and Eukaryotes :
Ribosomes
Prokaryotes: 70S
Eukaryotes: 80S and in mitochondria 70S
Examples for Chemoprophylaxis:
Antibiotics to prevent _________ of infections (e.g. UTIs).
Examples for Chemoprophylaxis:
Antibiotics to prevent recurrence of infections (e.g. UTIs).
Side effects of Aminoglycosides
Neurotoxicity
Oto- and nephrotoxicity
Significance of Microbiology
Oxygenic Photosynthesis
Occurs in plants, Algae and Cyanobacteria. It is the type of Photosynthesis that results in production of O2 in the atmosphere.
3 examples for Bacteria that are not identified with gram stain in labs?
Mycobacteria - mycolic acid do not absorbs gram stain.
Myoplasma - lack cell wall
Spirochaetes - too thin wall
In which infections will there be use of Passive artificial Immunity?
Diphtheria, Tetanus, Measles, HepB and Rabies
Also for Botulism Toxin (and other Insects and Reptile toxins).
Detection of Spores: Preparation process and Image
ירוקים מקנאה כי אני מלחיץ רק על ספורט ומים נקיים לספרנים החומים
- Malachite Green (Primary)
- Distilled water (Dicalorozation)
- Safranin (Counter)
- Endospore green and Other cells brown (Safranin)
Mechanism of evasion - Avoiding contact with Phagocytes:
Some pathogens _____ ______ an overwhelming inflammatory response which is _____ __ _____ the phagocytic defenses.
Mechanism of evasion - Avoiding contact with Phagocytes:
Some pathogens avoid provoking an overwhelming inflammatory response which is required to focus the phagocytic defenses.
Chemoheterotrophs:
use chemical and organic compounds, such as glucose (all medically relevant bacteria).
Infection Transmission routes - Horizontal Spread
Direct vs. Indirect
Direct - Human/Animal to Human
Indirect - Objects/Environment to Humen
Bacterial resistance to Penicillin arises from which location within the gram negative bacterial wall?
Periplasm - Contains the Beta-Lactamases
3 Risks of Misusing antibiotics
Tissue damage
Allergy development
MDR development
Naturally acquired passive immunity from mothers milk IgG provides immunity up to _ months and for ___ ___ ___ ___ ___ .
Naturally acquired passive immunity from mothers milk IgG provides immunity up to 6 months and for any immunity the mother has.
_____ is a _______ and a _______ vaccine that contains _______ ______ and parts of H. Influenzae .
Hib is a conjugate and a subunit vaccine that contains diphtheria proteins as well and parts of H. Influenzae
Horizontal Gene Transfer: Sex Pilli interaction allowing for the transfer of Plasmid between two Bacteria - This is ________ .
Horizontal Gene Transfer: Sex Pilli interaction allowing for the transfer of Plasmid between two Bacteria - This is Conjugation .
Significance of Microbiology
Decomposition or Biodegradation
Results in the breakdown of complex organic materials to forms of carbon that can be used by other animals
Sterilization by Dry Heat: Optional Protocols
___C for _ Hours
___C for _ Hours
___C for _ Hours
Sterilization by Dry Heat: Optional Protocols
140C for 3 Hours
160C for 2 Hours
180C for 1 Hours
Examples for Industrial Use of Microbiology in Model Research
E. Coli and Yeasts have been used to study the cellular life for basic science research
Resistance Abbreviations:
MRSA
Resistance Abbreviations:
MRSA = Methicillin Resistant Staphylococcus Au.
Facultative anaerobes express Superoxide dismutase and Catalase but also more ______ Enzymes so they are able to ferment Sugars as the sole Energy providing source if required.
Facultative anaerobes express Superoxide dismutase and Catalase but also more Glycolytic Enzymes so they are able to ferment Sugars as the sole Energy providing source if required.
Streaking technique - Inoculation of Plate
measuring amount of bacteria
- spread 0,1ml to agar media with few streak on one side
- Rotate the Plate by 90 Degrees and streak again
- Repeat again the second step
Disinfection by Heat:
Pasteurization consists of heating fluid to __C for ___ Minutes, following by a ____.
Pasteurization consists of heating fluid to 62C for 30 Minutes, following by a rapid cooling.
Antibiotics Interfering with 50S
Chloramphenicol
Macrolides (Erythromycin)
Lincosamide (Clindamycin)
Streptogramins
Horizontal Gene Transfer: DNA release after bacterial death ultimately causing homologous recombination in a new bacteria - This is ________ .
Horizontal Gene Transfer: DNA release after bacterial death ultimately causing homologous recombination in a new bacteria - This is Transformation .
Disinfectants:
Chlorine (Cl2) gas- Action and Use
Forms hypochlorous acid (HClO), a strong oxidizing agent. Disinfect drinking water; general disinfectant
Steps in preparation and Fixation of bacterial smears:
Cleaning from fat
Flaming slide 5 times with bunsen burner
_______ Transduction happens when a certain part (toxins/VF) of the host chromosome is broken down and packaged in the phage. This requires a lysogenic cycle for ______ DNA uptake in the ______ .
Specialized Transduction happens when a certain part (toxins/VF) of the host chromosome is broken down and packaged in the phage. This requires a lysogenic cycle for bacterial DNA uptake in the Prophase.
Exotoxins - Superantigens, 2 Examples
TSST 1 - S.Aureus
SPeC and SPeA - S. Pyogenes
IgM Negative and IgG Positive - Time of Infection
Old Infection - Could be due Vaccination
Years
Examples for Use of Animals in the Lab for Microbial Investigation:
-Demonstration of bacteria’s exotoxin production – in vivo test
Römer test - C. Diphtheriae given to Guinea pigs with pre-exposure to Anti-Toxin
What are 2 Dissemination possibilities for bacteria that can harm the host?
Bacteremia and Toxemia
Serum Resistant bacteria naturally include Gram_ species since those have no outer membrane.
Serum Resistant bacteria naturally include Gram+ species since those have no outer membrane.
Mechanism of evasion - Avoiding contact with Phagocytes:
Invading regions such as _______ __ _______ and ______ _____ like skin that are not _______ by Phagocytes.
Mechanism of evasion - Avoiding contact with Phagocytes:
Invading regions such as lumen of glands and surface tissues like skin that are not patrolled by Phagocytes.
Examples for Chemoprophylaxis:
NSAID therapy for familial _______ _______ showed regression of polyps.
Examples for Chemoprophylaxis:
NSAID therapy for familial adenomatous polyposis showed regression of polyps.
Examples for Chemoprophylaxis:
used to eliminate the carriage of __ ________ type b in children at high risk for disease (children <2 years in a family or day-care center where systemic disease is
documented)
Examples for Chemoprophylaxis:
used to eliminate the carriage of H. influenzae type b in children at high risk for disease (children <2 years in a family or day-care center where systemic disease is
documented)
Examples for Use of Animals in the Lab for Microbial Investigation:
-Propagation and maintenance of obligate intracellular bacteria
T.Pallidum growth in rabbits testis
What is present in a blood agar plate ?
Why?
5 -10 % of horses or sheep blood
Isolation of fastidious organisms and detect hemolysis
Aerobic Culture:
-Demands
Aerobic Culture:
37C, High Humidity, Normal Air and 1 Atm
Examples for Chemoprophylaxis:
Antibiotic prophylaxis to limit the spread of _________.
Examples for Chemoprophylaxis:
Antibiotic prophylaxis to limit the spread of epidemics.
3 factors effecting the disinfection quality:
1 - Initial number of Bacteria
2 - Time (at least 1 minute)
3 -Detergent Concentration
Quaternary ammonium compounds - Action and Use
Disrupts cell membranes. Skin antiseptics and
disinfectants.
History of Microbiology - Paul Ehrlich
1909
Modern Immunology Founder Precursor Technique to Gram Staining Chemotherapeutic Index Treatment for Syphilis Diphtheria Antitoxin Theory
Obligate intracellular parasites - Culturing:
Mycobacterium leprae
Can be grown in the mouse footpad or in the armadillo.
Definition of Pathogenicity
Ability of an organism to cause harm to its host. Represents a genetic component of the organism. It is manifested through its Virulence and Host interaction.
4 Stages of Infection
Incubation
Prodormial
Acute
Reconvalescence
History of Microbiology - Edward Jenner
1796
First vaccine created by him, It was for Small pox.
Derived from cow pox
What is this Vaccine for? and When is it recommended?:
DPTa
(Extra knowledge)
DPTa - Diphtheria, Tetanus and Pertussis
2/4/6 Months, 18 Months, 6 years
+ dTap Reactivation in 11 years
LPS - Effect of Macrophages:
Receptor and Products released with consequence
CD14
IL1, TNF and NO - Causing Fever and BP↓
Beta lactamase activity:
Digestion of penicillin
Significance of Microbiology
CO2 Fixation
Conversion process of inorganic carbon to organic carbon. Planktonic algae and Cyanobacteria (Later consumed by other animals)
________ agents are ones that correspond to the MIC value and _______ agents are the ones that correspond to the MBC Value.
Bacteriostatic agents are ones that correspond to the MIC value and Bactericidal agents are the ones that correspond to the MBC.
History of Microbiology - Joesph Lister
1867
Disinfection with Phenols
Modern Surgery
What are examples for molecules used for masking by bacteria?
Fibrin,Fibrinogen,Fibronectin,Collagen,Elastin
What are the parts of bacterial growth curve?
Lag phase
Log phase
Stationary phase
Death phase
Sterilization - Irradiation
UV - gamma
DNA damage
Determination of MIC - Punching test
1) Preparation of lawn culture (Flooded Equal Bacterial Growth Solid Media)
2) Making several holes on the surface (“punch”) and Pipetting Standard dilution of each Ab drug into them.
3) 37°C overnight
4) Measuring the diameter of the zone of inhibition
What is the Magnification power difference between Electron microscope and Light Microscope? What is the Microbiological Use of each?
Light - 250 nm , for Bacteria, Fungi and Protozoa
Electron - 0,1 nm, for Viruses
What is the key bacterial structure for UTI virulence?
Adhesins (on Fimbriae /Pili)
Comparison of Prokaryotes and Eukaryotes:
Cytoplasmic Membrane
Prokaryotes: Cytoplasmic membrane contains Hopanoids and Bioenergetics are associated with it.
Eukaryotes: Cytoskeleton-Cytoplasmic Membrane contains steroids (Cholesterol).
F plasmid
Mostly code for production of sex pili, when the male
cell extends his sex pili and one attaches to the female. This is a temporary cytoplasmic bridge allows the replicating F plasmid to cross and result is two male cells
Autotrophic bacteria
Use inorganic precursors to build their organic molecules
Antiseptics:
Isopropanol (50-70%) - Action and Use
Denaturation of Proteins and Solubilization of Lipids.
On Skin.
Infection Transmission routes - Vertical Spread
Congenital, Perinatal and Postnatal - Examples
1) Congenital - Through Placenta; HIV, CMV, Rubella
2) Perinatal - During Delivery; Chlamydia, E.Coli
3) Postnatal - Milk, Saliva, Contact; HIV.CMV
Which bacteria is detected by Slide agglutination ?
How does it work?
Qualitative or Quantitative ?
E. Coli Serotypes Detection, E.g. O157:H7
Latex agglutination of the released antigen
Qualitative: Y/N (15 tests instead of 100 method)
Chemoprophylaxis - Definition
Administration of an antimicrobial agent for preventing an infection or for suppressing contacted infection, before the clinical manifestations.
Resistance Abbreviations:
VRSA
Resistance Abbreviations:
VRSA = Vancomycin Resistant Staphylococcus Aureus
Torch test - when is it done ?
Before or in the First Trimester of the Pregnancy
Toxoid Vaccines (Disabled Virulence): 3 Examples
Toxoid Vaccines (Disabled Virulence):
1) C. Diphtheria
2) C. Tetani
3) C. Botulinum
What is this Vaccine for? and When is it recommended?:
HepB
(Extra knowledge)
Hepatitis B
12 years
Phenol coefficient
Dilution of the test compound that kills the organism in 10 min devided by Dilution of phenol compound that kills the organism in 10 min.
(appropriate Incubations and colonies count measured before)
Elimination of Prions - Protocol:
Immerse 2N ____ for 1 Hour / 1N ____ for 2 Hours
Autoclave at ___C, __ ATM, for 1 Hour
Two steps should be________!
Elimination of Prions - Protocol:
Immerse 2N NaOH for 1 Hour / 1N NaOH for 2 Hours
Autoclave at 134C, +2 ATM, for 1 Hour
Two steps should be repeated one more time!
Liquid media for Anaerobic culture - Methods of Environment Production for Anaerobes
Before inoculation, O2 is eliminated from media by means of boiling. After inoculation, media are sealed by means of sterile wax or Vaseline.
Which molecule is mimicked by penicillin?
Why?
D-Ala-D-Ala (crossed linked NAM by transpeptidase forming Peptidoglycan)
This is a beta-Lactam ring which is part of peptidoglycan.
When penicillin binds the active site (competitive inhibition) and leads to ultimately membrane weakening.
What is this Vaccine for? and When is it recommended?:
IPV
(Extra knowledge)
IPV - Poliovirus
2/4/6 Months, 18 Months, 6 years
On which Bacterial spectrum will we be able to find Endotoxins?
Gram Negative
Obligate intracellular parasites - Culturing:
Coxiella burnetii
Coxiella burnetii must be done in a biosafety level 3 containment facility (Highly Infectious)
What are the 4 general different mechanisms of action of antibiotics:
Inhibition of: Cell wall synthesis Proteins synthesis DNA synthesis or Membrane permeability changes
How do Anionic Detergents work?
gross disruption of the lipoprotein framework of the cell
membrane.
4 ECM Enzymes with Antiphagocytic Effect for Bacteria
Leucodin (Pore Forming Toxin)
Coagulase (S.Aureus)
Hemolysins (S.Aureus, S.Pyogenes)
Proteases (Legionella)
Antibiotics altering DNA and RNA synthesis
Quinolone
Metronidazole
Rifampin
Principles of Antibacterial Chemotherapy -
Dosage Principle and Factors to Consider
Antibiotics should be given allowing for seum level higher than MIC at the locus of Infection. Factors: Age, Weight, Pregnancy, Liver and Kidney Function.
(Timing and Method of Uptake and Price as well)
Examples for Industrial Use of Microbiology in Pharmaceutical Therapy
Antibiotics - Bacillus Subtilis yields Bacitracin
Vaccines - Version of Diphtheria Toxin are used as Toxoid from Immunization
Antiseptics:
Tincture of Iodine (2% I2 in 70% alcohol) - Action and Use
Inactivates proteins
on skin and In drinking Water
Vital Staining: Preparation process and Purpose
- Wet mount preparation
- Drop of Methylene Blue
- After ten minutes low power magnification
- Fast detection by higher contrast of Neisseria (example)
ID50/LD50/TCID50 Measurements Gave a value more than 10^5 Germs.
What does it mean?
Low Virulence since this is a High Germ count that causes 50% of Hosts/Cultures to have a pathologic conditions (Infection/Death/Damage).
SIRS
Systemic Inflammatory Response Syndrome (Sepsis)
Comparison of Prokaryotes and Eukaryotes :
Cell Division
Prokaryotes: Binary fission (Transfer via conjugation, transduction and transformation)
Eukaryotes: Mitosis
Determination of MIC - Tube Dilution (“Macro-“)
1) Sequential dilution of the drug
2) Equal amount of bacteria into each tube
3) Incubation Overnight at 37 C
4) MIC will be the Highest dilution tube that’s transparent!
Components of Peptidoglycan?
Arrangement?
NAG - N-Acetylglucosamin
NAM - N-Acetylmuramate
3-5 Amino acids peptides
Crosslinked in sheets, Peptides bind only NAM
Examples for Use of Animals in the Lab for Microbial Investigation:
Producing antibodies for passive immunization
Antibodies for Botulism Toxin Produced in Mice.
Fluroquinonolons side effect
Tendon and cartilage damage
A patient with a splenectomy history has increased susceptibility to bacteria with which component?
Capsule - Polysaccharide Coat is recognized by Splenic Macrophages
TCID50
A Quantitative Determinant of Virulence
Tissue Culture Infecting Dose - Number of Germs damaging 50% of Cultures.
Basic Structure of Antibody
2 Heavy chains 2 light chains Disulfide bonds Variable region - Antigen Binding Fab Constant - (Crystallizable) Fc
IgM and IgG are both Negative - What is the time of the Infection
Too early or No Infection
Could be early days, Pathogen would be a better way for detection at this point
Examples for Use of Animals in the Lab for Microbial Investigation:
-Determination of virulence
The LD50 (also LD90) is a laboratory measurement of virulence, which determines the number of microbes necessary to kill 50% (or 90%) of the animals infected.
Transport swab:
Example
Components
Transport swab: Stuart Transport (Just keeping the microbe alive) Thioglycolic Acid (Reduction Agent) and Electrolytes
What does the Spore Cortex consists of ?
Modified Peptidoglycan Layer
Disinfection by Heat:
Examples for Bacteria that will survive Pasteurisation
Disinfection by Heat: Pasteurisation Survivors
Spore Forming Bacteria like B. Anthracis and M. Tuberculosis
Colonization is ____ multiplication of microbes at the site of ___ or Close to it.
Colonization is Irreversible multiplication of microbes at the site of entry or Close to it.
Microaerophilic Bacteria Express only _____ and this allows them to survive in low amount of Oxygen while mainly relying on ______.
Microaerophilic Bacteria Express only Superoxide Dismutase and this allows them to survive in low amount of Oxygen while mainly relying on Fermentation.
Vancomycin is an example for a non ____-______ antibiotic that acts on the bacterial ____ ____ .
Vancomycin is an example for a non Beta-Lactam antibiotic that acts on the bacterial cell wall.
Disinfectants:
Mercuric chloride- Action and Use
Inactivates proteins by reacting with sulphide groups
Disinfectant, although occasionally used as an antiseptic on skin
What is the usual function of capsules and what are the possible components?
Antiphagocytic and Adhesion
Usually made of Polysaccharides but there are B. Anthracis with Poly-D-Glutamate Capsule and S.Pyogenes with Hyaluronic Acid Capsule.
_______ Transduction happens when the host chromosome is broken down and a random part of it is packaged in the phage with the Viral DNA.
Generalized Transduction happens when a random part of the host chromosome is broken down and packaged in the phage with the Viral DNA.
Difference between antibiotics and chemotherapeutics?
Antibiotics - produced by microbes
Chemotherapeutics - produced synthetically
What is the importance of extracellular enzymes found on bacteria? (Collagenases, haemolysin..)
In vitro - diagnostics
In vivo - virulence factors
Pharmaceutical - antibiotics
________ are substances produced by pathogens that cause damage to phagocytes e.g. __________ or ______ released before or after phagocytosis.
Aggressins are substances produced by pathogens that cause damage to phagocytes - Extracellular enzymes or toxins released before or after phagocytosis.
Classification of Exotoxins based on toxicity mechanism.
Intracellularly-acting toxins - 3 Examples
(AB toxins: A-enzymatically active, B for binding )
- Inhibition of protein synthesis (Diphtheria)
- Growth inhibitors (LF - Anthrax MAPK Cleavage)
Study (monitoring) of antimicrobial drug level in clinical samples
Determination of MIC - Punching test is done twice in parallel (Holes on lawn Culture): one for calibrated bacterium that is pre-incubated and second from the clinical sample (Serum)→diameter of the zone of inhibition is compared !
IgM Positive and IgG Negative - Infection Time
Acute Infection
(Pathogen Should be present and observable too, Days or Weeks)
This could also be a False Positive IgM!
What is the type of enrichment required for Listeria?
Cold Enrichment of the Culture
Determination of MIC - Agar plate dilution test
1) Various concentrations of antibiotic are incorporated in a series of plates and Agar solidify them
2) Equal amount of bacteria into each Agar
3) Incubation Overnight at 37 C
4) MIC will be the Highest dilution tube that’s transparent!
What are Neisser I. and Neisser II stain contents?
methylene blue (I) and crystal violet (II)
What is this Vaccine for? and When is it recommended?:
HiB
HiB - H. Influenzae B
2/6 Months, 18 Months
IgM and IgG are Positive - Time of Infection
Recent or Acute Infection
3 weeks to Months
Phenetic definition of Phylum
Group of Organisms with a certain degree of morphological or developmental similarity.
______ is a _____ vaccine that has 2 _______ components for Diphtheria and Tetanus and 1 _______ component for Pertussis.
DPT is a triple vaccine that has 2 toxoid components for Diphtheria and Tetanus and 1 Inactivated component for Pertussis.
Western Blot - Ab differentiation and a better alternative
Larger Ab will move a further distance .
Line Immunoassay could be a replacement.
Flagella stains material
“Titanic Flagella”
Tannic Acid
Tube agglutination - For which bacteria?
Qualitative or Quantitative?
Salmonella typhoid (Grubel-Widal Reaction)
Qualitative - Specific Antibodies from serum
Quantitative - Antibody Titer Determination
Examples for Use of Animals in the Lab for Microbial Investigation:
Producing monoclonal antibodies for serological tests-
Hybridoma method in mouse
Inactivated Vaccines (Cultured and Destroyed):
3 Examples
(Extra knowledge)
Inactivated Vaccines (Cultured and Destroyed):
1) V. Cholera
2) B. Pertussis
3) Y. Pestis (Bubonic Plague)
What amino acid abundance with water contributes the most for the initiation of the germination of a spore to bacterial cell?
Alanine
Give Examples for a Low Therapeutic Index and High Therapeutic Index Antibiotics:
Low Therapeutic Index - Aminoglycosides
High Therapeutic Index - Penicillin
Antibiotic produced by Bacillus subtilis:
- Nystatin
- Streptomycin
- Amphotericin
- Bacitracin
- Gentamicin
Bacitracin
When does MBC is important for the determining antibiotic amount of prescription? Which conditions?
Severe cases of infection:
Osteomyelitis, Endocarditis, Sepsis, Immunocompromised
_____ is a _______ and a _______ vaccine that contains _______ ______ as well and it contains ______ of S. Pneumoniae.
PCV13 is a conjugate and a subunit vaccine that contains diphtheria proteins as well and it contains subunits of S. Pneumoniae.
How does Gram staining shows on both kinds of bacteria?
Gram+ : purple
Gram- : pink/red
Definition of Virulence
Degree of pathology caused by the organism.
depends on its Virulence factors : Structural as cell wall or Capsule and Biochemical as Exotoxin or Endotoxin.
Torch test - Microbes Initials meaning
Toxoplasma Other - Syphilis,VZV, S. Agalactiae Rubella CMV Herpes
What are the two time points when Antigenic variations may take place?
1) Between infections of the pathogen
2) During the course of Infection
Examples for Chemoprophylaxis:
Very effective against ____ signs of tuberculosis
Examples for Chemoprophylaxis:
Very effective against early signs of tuberculosis
Glycopeptides antibiotics
Vancomycin and Teicoplanin
MIC - for Classification of Antibiotics
Definition and Unit
Minimal Inhibitory Concentration that prevents Growth of the Microbe in the Host ant relays on the Host defence to finish the Job. (ug/ml or mg/L)
Ozone- Action and Use
Generates lethal oxygen radicals.
Purification of water, sewage.
Equivalence Zone
The right amount of Ag in serum that provides the Maximal level of agglutination
What is present in a chocolate agar plate?
Why?
Lysed RBCs that have been heated to 80c.
Growing of Fastidious bacteria such as Haemophillus Influenzae
Definition of Infection
Invasion and Multiplication of Microorganisms that are not natural to the body or area.
Risks of Using Antibiotics: Inhibition of
immune system
- Embryotoxic action
- Formation of drug resistance
Serum Resistant bacteria the include Gram_ species that cause bacteremia .
Serum Resistant bacteria the include Gram- species that cause bacteremia .
How can we prove true motility with direct examination in microscope? Otherwise, what is observed (could be perceived as motility)?
Observing a bacteria swimming from one side of the slide to the other. Otherwise it is just brownian motion.
MBL - Initials
MetalloBeta Lactamases
MIC - Minimal inhibitory concentration
Methods of Determination: (3)
Micro and Macro Dilution
Agar Dilution
E-Test
Examples for Chemoprophylaxis:
Antibiotic prophylaxis for _________________ people to prevent opportunistic infections.
Examples for Chemoprophylaxis:
Antibiotic prophylaxis for immunocompromised people to prevent opportunistic infections.
Antimicrobial Drugs in combination - Possible Outcomes
1) Indifference
2) Antagonism
3) Synergism
Key Components of Gram+ Bacteria cell wall
Lipoteichoic acid (LTA) Peptidoglycan (much thicker with it)
What kinds of different consistencies can culture media have?
Broth - liquid media
Solid media
Semisolid media
Toxoid containing:
- BCG vaccine
- DPT vaccine
- Pneumococcal 13 valent vaccine
- Meningococcal polyvalent vaccine
- Haemophilus influenzae vaccine
DPT vaccine
Obligate anaerobe infect while usually causing _____ in the abdominal cavity and ______ in lungs in order to form a good environment for themselves.
Obligate anaerobe infect while usually causing abscess
in the abdominal cavity and aspirations in lungs in order to form a good environment for themselves.
What 2 types of Bacteria can form spores?
Aerobic Bacillus and the Anaerobic Clostridium.
Conjugate Vaccines :Conjugation of Bacterial _____ to proteins (e.g Toxins created enhanced Immune recognition of the ______.
Conjugation of Bacterial Polysaccharides to proteins (e.g Toxins created enhanced recognition of the Polysaccharides . Example - H. Influenzae B
Mechanism of evasion - Inhibition of phagocytic engulfment:
Resistance to phagocytic ingestion is usually due to a _______ of the bacterial ________, ______ or a _______ .
Mechanism of evasion - Inhibition of phagocytic engulfment:
Resistance to phagocytic ingestion is usually due to a component of the bacterial fimbriae or a capsules.
Microaerophilic Culture:
- Oxygen and Carbon Dioxide Percentage Needed
- Example
Microaerophilic Culture:
- Oxygen 10% and CO2 10%
- Skirrow Medium for H.Pylori and Campylobacter
What is the Microscopic activity of Spores?
Refreactive
Precautions in case of danger of cholera, EXCEPT:
- Providing sterile drinking water
- Passive immunisation
- Safe sewage removal/processing
- Increasing personal hygiene
- Providing effective therapeutic measures
Passive immunisation
Capsules, Outer membrane permeability barrier of Gram Negative Bacteria and LPS are all examples of ____ Mechanisms ____ ____ _____.
Capsules, Outer membrane permeability barrier of Gram Negative Bacteria and LPS are all examples of Evasion Mechanisms from host defences.
What are the 2 structures of Spores that allow survival in Chemical and Heat changes?
1) “Keratin-Like” Protein Core- Protecting from Chemicals
2) Dipicolinic Acid Coat - Protects from Heat
Comparison of Prokaryotes and Eukaryotes :
Membrane enclosed organelles
Prokaryotes - None
Eukaryotes - Lysosomes, Golgi, ER, Mitochondria
ESBL - Initials
Extended Spectrum Beta Lactamases
Order of Taxonomy:
Kingdom→ _______→ Class → Order → _______→ ______ → Species
Order of Taxonomy:
Kingdom → Phylum → Class → Order → Family → Genus → Species
Exotoxins - Inhibition of Protein Synthesis, 2 Examples
1) ADP Ribosylation - E2F Inactivation by Diphtheria’s lethal toxin and Exotoxin A of Pseudomona
2) Shiga and Shiga-like (EHEC) toxins binding 60S
Antiseptics: Silver nitrate (AgNO3) - Action and Use
Precipitates proteins.
General antiseptic and used in the eyes of new-borns.
Welch Method - Preparation process and Purpose
וולץ מסניף קריסטל ,מחלל את חם, כופר ומסלף דברים העיקר לראות שמיים כחול בהיר, חי בקפסולה הבנאדם
- Hot crystal violet
- wash with copper-sulfate
- Capsule appears pale blue
A persistent, asymptomatic after treatment, bacterial colonization of the trachea of a CF patient could be caused by _____ properties of a bacteria.
A persistent, asymptomatic after treatment, bacterial colonization of the trachea of a CF patient could be caused by Biofilm properties of the bacteria.
Significance of Microbiology
Nitrogen Fixation
Some Bacteria remove N2 from the atmosphere and converts it to Ammonia for use by plants and animals.
What are disinfectants and Antiseptics?
Disinfectant - toxic, for inorganic surface
Antiseptics - less toxic for organic surface
MBC - for Classification of Antibiotics
Definition and Unit
Minimal bactericidal concentration of the antibiotic.
lowest concentration of an antibiotic that kill 99.9% of a certain (ug/ml or mg/L)
Ziehl-Neelsen Staining (Acid Fast): Preparation process and Purpose
נילסן אכל קבבול בפושקין התעצבן שלוש פעמים אז שפכו עליו מים והקיא את כל ה96% אלכוהול נהיה חיוור אז שמו לו מטלית כחול
- Carbol-Fuchsin (Mycolic acid binds)
- Heat 3 times and Wash with water
- Decolorization with 96% alcohol
- Counter stain with Methylene Blue
What is Natural resistance?
A quality of the target bacteria as in contains enzymes for elimination of the antibiotics or lacking the target molecule for the antibiotics attack.
What is this Vaccine for? and When is it recommended?:
MMR
(Extra knowledge)
MMR - Measles, Mumps, Rubella
15 Months + Reactivation in 11 years
What componants solidify the culture?
Agar or gelatin
When there is a patient with food poisoning he will receive Passive Immunization with IgG through injection to prevent the risk of ___ such as ____.
When there is a patient with food poisoning he will receive Passive Immunization with IgG through injection to prevent the risk of toxins such as Botulism.
Disinfectant:
Formaldehyde (8%) - Action and Use
Reacts with NH2, SH and COOH groups
Kills Endospores.
When would we use combinations of antibiotics
Synergistic effect
Increased length of use like in TB
High variance of resistance or types of microbes
Gas-pack jar (Compartment) - Methods of Environment Production for Anaerobes
Cultivation of Anaerobic microbes by - Placing a Gas Pack in the Jar that releases H2 and CO2, a Catalyst in the cell produces Water from O2 and H2 thus ultimately eliminating the Oxygen in the sealed Jar.
What is the cause of the disinfectant effect of Agents such as Formaldehyde or Ethylene Oxide?
Alkylating Proteins
Agglutination
Ag-Ab reaction linked to cell surface - Visible to the naked eye
Examples for Chemoprophylaxis:
Prophylactic antibiotics prior to dental work for individuals with damaged ____ _____.
Examples for Chemoprophylaxis:
Prophylactic antibiotics prior to dental work for individuals with damage heart valves.
History of Microbiology - Robert Koch
1880
Founder of Modern Bacteriology
Identified the specific causative agents for Tuberculosis cholera and Anthrax.
Koch’s Postulates - 4 criteria designed to establish a cause effect between microbe and disease
Anaerostate (Compartment) - Methods of Environment Production for Anaerobes
Cultivation of Anaerobic microbes by - Air Expulsion by Pressure Cylinder, Air Aspiration and Filling or Anaerocult that produces the Correct Gas mixture.
Biological method - Fortner-technique: Environment Production for Anaerobes
Growing Anaerobes and Aerobes together on Blood agar - Anaerobe over consume the Oxygen allowing for an Anaerobic environment for the other Anaerobe.
Generalized transduction
Virus is transmitting the Genetic matter to a bacterium, which multiples and lead to production of more viral proteins and DNA.
Chemical method - Liquid media for Anaerobic culture
Thioglycolate broth allows for reduction of Oxygen to water in the tube. Aerobes will culture the top of the liquid (Contact with air) anaerobe the bottom.
Mechanism of evasion - Survival inside phagocytes:
Inhibition of _______________________ fusion - This allows for no _________ discharge inside the phagosome.
Mechanism of evasion - Survival inside phagocytes:
Inhibition of Phagosome-Lysosome fusion - This allows for no lysosomal discharge inside the phagosome.
Antibiotics Interfering with 30S
Aminoglycosides - Gentamycin, Streptomycin
Tetracyclines - Oxy-Tetracycline and Doxycycline
When are endotoxins released from a bacteria?
Cell Death
Wet Mount: Preparation process and Purpose
- drop of bacterial suspension in the middle of slide
- Coverslip - Surface tension holds the sample
- 40x magnification
Resistance Abbreviations:
VRE
Resistance Abbreviations:
VRE = Vancomycin Resistant Enterococcus
Examples for enzymes aiding in killing phagocytes before phagocytosis
Hemolysin - Lyse RBCs as well
Streptolysin O - Lysosomal granules explosion in cell
Comparison of Prokaryotes and Eukaryotes : Glycocalyx
Prokaryotes: Present as capsule or slime layer
Eukaryotes: Present in some cells that lack a cell wall (Animal cellls)
Monitoring of antibiotics (prevention of kidney failure)
Calibration curve of weekly taken cultures and their relative inhibition zones
Control of Bacterial Sterility of Parenteral Drugs
Thioglycollate Medium - Parallelly under Aerobic and Anaerobic Conditions at 37C for 7 days
Tetracyclines side effect
Complex with Calcium colorize and defect enamel.
Discoverer of Penicillin:
- Paul Ehrlich
- Selman Waksman
- Robert Koch
- Alexander Fleming
- Ian Fleming
Alexander Fleming
When there is a patient with animal bites he will receive Passive IgG through injection to to prevent the risk of ___.
When there is a patient with animal bites he will receive Passive IgG through injection to to prevent the risk of Rabies.
LPS - Effect on Complement:
Products released with consequence
C3a - BP↓ and Edema
C5a - Neutrophills Chemotaxis and Edema
What is the Utility of having a Glycocalyx on the bacterial Surface?
Aids in formation of Biofilms and so survival in harsh conditions is easier e.g. S.Epidermidis in Catheters
Sterilisation - Mechanical
Bacteria filter
0.45 or 0.1 microns pores
(Mycoplasma or viruses can pass)
What do culture media actually contain?
Carbons Source (Sugar), minerals, buffer, yeast extraction, water and Amine Source (Casein derivatives Tryptone/Peptone)
Which of the following causes the purple color staining in Gram positive Bacteria: 1)LTA B)NAM C)Capsule D)Fimbria
NAM
Rifamycin side effects
Hepatitis - Orange body fluids
Pros and Cons of Detergents?
Pro:low toxicity levels, lack of smell, good skin tolerance
Con:Ineffective for Gram Negative Bugs
What kinds of specificity can culture media have?
Give examples
(4 types in total)
Basic media - blood media (5%)
Selective media - Chocolate media with Vancomycin
Differential media - EMB, Eosin Methylene Blue
Special Media - Chocolate media
Definition of Antibiogram
The Spectrum of Antibiotic options to be given in a situation
What is this Vaccine for? and When is it recommended?:
PCV13
(Extra knowledge)
PCV13 - S. Pneumoniae
12 Months
Pure culture
After streaking of mixed bacteria with special streaking pattern distinct, isolated colony can be grown on the surface of culture media. Every bacterium shows the same biochemical pattern, and antibacterial sensitivity pattern, belongs to the same species. Preparation of pure culture is essential to the identification, and other microbiological investigation.
Phenolic compounds - Action and Use
Denature proteins and disrupt cell membranes.
Antiseptics at low concentrations; disinfectants at high concentrations
Bacterial Antigens names for Capsule, Cell Wall, Flagella, Fimbria
Capsule - K antigen
Cell wall - O Antigen
Flagella - H Antigen
Fimbria - F Antigen
Mechanisms of Evading immunological response:
3 examples for Tolerance formation to Bacterial Ag
- Fetal exposure to the antigen
- High persistent doses of the circulating antigen
- Molecular Mimicry
What specimens are detected by Dark Field Microscopy?
Spirochaetes - Treponema Pallidum, Borrelia Burgdorferi, Leptospira Icterohaemorrhagiae
Invasin
Extracellular Protein binds to Integrin receptor allowing for Bacteria entry to Epithelial cells
Bacterial Isolate
a population of bacteria that belong to one species, and were isolated at a certain time from a certain origin.
MODS
Multi-organ dysfunction syndrome result of shock (in our case septic shock)
Comparison of Prokaryotes and Eukaryotes :
Fllagela
Prokaryotes: 2 Protein building blocks - flagellin
Eukaryotes: Complex (9+2) Microtubules structure
Types of metabolism preformed by aerobic and anaerobic bacteria?
Aerobic bacteria - Respiration
Anaerobic bacteria - Fermentation
What is an Endoflagella?
Where is it found?
Flagella located in the cytoplasm
Usually in Spirochetes
Mechanisms of Bacteria Evading Complement:
Alterations of LPS - 2 Examples
1) LPS O antigen with Sialic Acid prevents the C3 convertase formation. (N. Meningitidis and H.Influenzae).
2) LPS with long intact O antigen side chains causes distancing of MAC from Bacterial Membrane
What are the two functions of Eosin Methylene Blue agar medium?
- Selective: for Isolation of Gram Negative
- Differential: for Isolation of Lactose Fermenting
Ideal Antibiotic Drug Factors (6)
Selective Toxicity -Cidal/-Static Reach the target +good concentration active (Kinetics) Spectrum Lack of Side effects Little Resistance development
3 Desired effects of Antimicrobial Drugs in combination
When applied for the correct pathogen
1) Synergism for Strong Pathogen/Weak Immune Sys
2) Extend Spectrum for Empirical Identification of Cause
3) Prevent Induction of Resistance Mechanisms for Long treatments (E.g Tuberculosis - R.I.P.E)
What is the cause of the disinfectant effect of Agents such as Iodine , Chlorine or Hydrogen Peroxide?
Oxidizing Proteins
3 ECM Enzymes Facilitating Bacterial Invasion
Streptokinase (S.Pyogenes)
Collagenases (Clostridium)
Hyaluronidase (S.Aureus, C.Perfringens, T.Pallidum)
Disinfectant catagories;
Agents against cell wall Detergents Peptide denaturating agents Agent acting on nucleic acids Oxidazing agents Alkylating agents
2 Examples for a Polypeptide antibiotics
Bacitracin and Polymyxin
Flocculation
Large Precipitation
Used for Syphillis detection
____ _____ : Antigenic determinants of a bacterium are so closely related chemically to host “self” components that the immune system cannot recognize it.
Molecular Mimicry : Antigenic determinants of a bacterium are so closely related chemically to host “self” components that the immune system cannot recognize it.
What is the advantage of Transport plates over transport swabs? Examples
Saves time by letting the bacteria grow meanwhile (Culture). Uricult and Gono-line.
Mechanism of evasion - Survival inside phagocytes:
Survival inside the Phagolysosome due to components of the bacteria or Extracellular substances produced that resist the __________ _____________ .
Mechanism of evasion - Survival inside phagocytes:
Survival inside the Phagolysosome due to components of the bacteria or Extracellular substances produced that resist the lysosomal constituents .
Resistance Abbreviations:
VISA
Resistance Abbreviations:
VISA= Vancomycin Intermediate Staphylococcus Aureus
Which could serve as a strong Antigen, Bacterial Lipid or Protein ?
Protein
Narrow spectrum antibiotics (2):
- Penicillin
- Tetracycline
- Chloramphenicol
- Rifampicin
- Streptomycin
Penicillin and Streptomycin
Determination of MIC - Microdilution test
Equal amount of bacteria into each
1) Sequential dilution:96-well polystyrene microtiter plate
2) MIC will be the Highest dilution tube that’s transparent!
Selective Toxicity (Substance)
A Substance should be harmful to the pathogen but not harmful to host.
Koch’s Postulate - Criteria 3:
The Microorganism should ___ ___ when introduced into ____ ____ .
Koch’s Postulate - Criteria 3:
The Microorganism should cause a disease when introduced into healthy organisms .
Limitations of Chemoprophylaxis:
- Risk of side effects, Like Dysbacteriosis
- Possible High Cost
- Possible Low Incidence of the disease
Paratrophic organisms
Need a living host for reproduction
LAL test
Atlantic horseshoe crab blood aggregates in presence of Lipid A - Detection of Pathogens
Neisser Staining: Preparation process and Purpose
ניסר לו איזה תירס עם וולט וחצי כדי לעבוד על כלי-סידן ובלוטין
- Stain fixed smear of Corynebacteria with mixture of N.1 and N.2 (2:1 ratio)
- Water Wash
- Chrysoidine dye then Dry with blotting paper
- Detection of Volutin Granules
How can we eliminate resistant Spores?
Soap and water washing
Not alcohol
Artificial Snow, Botox, Pesticides, Hyaluronic Acid are all examples for ________ _________ used in the unusual Industrial ways.
Artificial Snow, Botox, Pesticides, Hyaluronic Acid are all examples for Bacterial Products used in the unusual Industrial ways.
7 conditions to keep in mind when preparing a bacterial culture
- Nutrients
- Temperature
- Oxygen - Aerobic/Anaerobic/microaero/capnophilic
- Water
- pH 7.2-7.4
- Osmotic pressure (NaCl concentration)
- Sterile environment
History of Microbiology - Dmitri Ivanovsky
1892
Discoverer of filterable nature of viruses - founder of virology.
Discovered the Tobacco mosaic Virus.
Classification of Exotoxins based on Site of Action
Neurotoxins - Botulinum Cytotoxin - Pertussis Toxin Enterotoxin - Cholera Toxin Leukocidin - WBC Pore forming Hemolysin - RBC Pore forming
Koch’s Postulate - Criteria 4:
The Microorganism must be ___ from the ____ ___ ____ and Identified as being Identical to the ____ ___ ___.
Koch’s Postulate - Criteria 4:
The Microorganism must be Re-isolated from the Experimental Diseased Host and Identified as being Identical to the Original causative agent.
3 Options for Adhesion to Host cells by Bacteria
1) Fimbrial Glycolipid has a Glycoprotein Interaction
2) Bacterial Adhesion Molecules bind Glycoprotein (Afimbrial)
3) Hydrophobic Adhesion: Bacterial Cell wall
ID50
A Quantitative Determinant of Virulence
Infective Dose - Number of Germs inducing disease in 50% of Hosts.
Principles of Antibacterial Chemotherapy -
Toxemia as a treatment Consequence
(Jarisch–Herxheimer reaction)
Bactericidal Antibiotics cause great number of Bacterial death, at the beginning of the treatment large amount of toxins are released. Prevention of Shock is Essential.
Candidiasis
Candida (yeast) normally live harmlessly in our body
With antibiotic use, the normal flora in the vagina or oral cavity is disrupted → candida grows → fungal infection formed
Elek’s Test - Precipitation
Corynebacterium Diphtheriae Toxin Detection
The specific Toxin could be released and detected by the Filter Paper (bind to antitoxin)
History of Microbiology - Ignaz Philipp Semmelweis
1847
Early antiseptics Pioneer - “Savior of Mothers”
Chlorine hand wash prevented Puerperal Fever in Child Birth (Streptococcus Pyogenes)
India Ink and Quelling test are 2 Important tests that help in visualization of _________ bacteria.
India Ink and Quelling test are 2 Important tests that help in visualization of Encapsulated bacteria.
Obligate anaerobe lack oxygen protective enzymes and are mostly part of the ____ ____ and _____ Flora. They prefer living in _____ if formed.
Obligate anaerobe lack oxygen protective enzymes and are mostly part of the oral cavity and GI Flora. They prefer living in abscesses if formed.
Determination of MIC - E –test
Placing a plastic strip impregnated with the concentration gradient of the certain antibiotic on a Lawn Culture. From the scale you can read the MIC numerically. MIC = number where the elliptic zone crosses the plastic strip.
_______ Antibody formation can happen as a result of pathogen ability to present ______ antigenic determinants.
Ineffective Antibody formation can happen as a result of pathogen ability to present various antigenic determinants.
Phylogenetic definition of Phylum
Group of organisms with a certain degree of evolutionary relatedness.
Comparison of Prokaryotes and Eukaryotes :
Nucleus
Prokaryotes - Nucleoid with no Membrane or Nucleolus
Eukaryotes - Nucleus with Membrane and Nucleolus
Risks of Using Antibiotics: Penicillin
- 5% of people are allergic (hypersensitivity reaction type 1) to penicillin
- This can cause anaphylactic shock
(all other types of HSN are also Possible with it)
Quellung Reaction - Examples for target bacteria:
“KESHA”
Encapsulated Bacteria: (KESHA)
Klebsiella, E. Coli , S. Pneumoniae, H. Influenzae, (S.)Agalactiae, (Salmonella, N. Meningitidis).
Colony morphology - Example for Identification by Color
Pseudomona Aeruginosa - Pyocyanin and Pyoverdin
S. Aureus - Yellow
Serratia Marcescens- Red
Colony morphology - Example for Identification by Smell
Pseudomona Aeruginosa- Grape Like
Colony morphology - Example for Identification by Motility
Proteus Mirabilis - Swarming Motility
Enterobacter Cloacae - Highly Motile
Klebsiella Pneumoniae/Shigella Dysenteriae - Immotile
Colony morphology - Example for Identification by Hemolysis
Clostridium Perfringens - Double Zone α + β
Eosin-Methylene-Blue medium (EMB) - What happens when a lactose fermenting bacteria is introduced? Describe the Process
Lactose Fermentation → pH ↓→ eosin loses its color→ The blue color remains→ the bacteria incorporate it→ the colony is BLUE in color (Klebsiella Pneumoniae).
*E.Coli → Metallic Green!
Eosin-Methylene-Blue medium (EMB) - What happens when a non lactose fermenting bacteria is introduced? Describe the Process
No Lactose Fermentation → pH same→ Pink color is enhanced (Salmonella Typhi).
Russel culture media containing: Brilliant sugar= dextrose, saccharose, lactose. Andrade indicator- decolourised fuchsin: Acid = red
Possible Outcomes:
Lactose + dextrose fermentation = whole culture Red
Lactose + dextrose fermentation + gas production = Red
with Burst
Lactose -, dextrose +: the high part is red, the slant not
TSI (triple sugar iron agar): Glucose, Lactose, Sucrose, pH indicator, Iron.
Colors of: H2S production, Glucose is broken down, Lactose are broken down.
H2S production - Black, In Salmonella Typhi / Proteus
Glucose is broken down - Only Slope Yellow, Proteus
Lactose are broken down - All Yellow, Escherichia Coli
ONPG- The demonstration of the β - galactosidase enzyme
Determination of the ability of an organism to produce beta-galactosidase, that hydrolyzes ONPG to ortho-nitrophenol = Fast Lactose Fermenter
The ureum-indol (UI –medium) / Christensen medium
Contains: dextrose, ureum, phenol red indicator
Examination of the Tryptophanase enzyme- demonstration of the indole production with Kovács- reagent on UI- medium. Possible Outcomes:
1) Tryptophanase enzyme is cleaving tryptophan →free indole ring + pyruvic acid + ammonia.
2) If free indole ring is present, the aldehyde group containing indicator turns RED
Antibody Titer (Quantitative Tube Agglutination): Definition and Example
A serial dilution is made from patient sera, known antigen solution is added to each tube. The Result Value Corresponds to the highest dilution factor (lowest concentration) that still yields a positive reading. Grubel-Widal reaction - Salmonella typhi.
What Serological Measurement is described here? Antibodies or Ag in vitro are bound to the surface of latex particle or red blood cells so that the binding partner will cause agglutination. Modern, fast technique. The antibodies or the Antigens are in vitro bound to latex particle.
Latex Agglutination - Diagnosis of meningitis
Co-Agglutination - S. Aureus Diagnosis
(Both are Passive Processes)
What Serological Measurement is described here?
Precipitation ring can be seen in the equivalent zone- the zone where the amount of antigen and antibody are equal.Qualitative measurement.
Steps: introduce serum into the tube → add ag → disc-shaped zone of precipitation
Ring Precipitation - Liquid phase precipitation
What Serological Measurement is described here?
The precipitation reactions can be visualised by allowing the Ag and the Ab to diffuse towards each other through agar gel - Immunodiffusion.
Semi-solid phase precipitation (agar gel precipitation)
What Serological Measurement is described here?
-Filter paper impregnated with anti-toxin (antibody against bacterial toxin) is placed in agar plate.
- Bacterial strains to be tested are placed onto the surface of the plate at right angles to the paper strip.
- Antitoxin diffuses from the filter paper into the agar-gel, binds to the toxin produced by the bacteria.
- A fine precipitation line is formed in the equivalence
zone.
ELEK’s test - Diphtheria Toxin detection
Antituberculotic drugs: Common Characteristics
- All cause hepatotoxicity
- All are given orally for a period of 6-9 months
- High penetration into most tissues (must reach the center of caseous granuloma)
Antituberculotic drugs:
Phase I of treatment - 2 months (RIPE)
- Rifampin (Rif-AMP-in)
- Isoniazid (ISO-niazid)
- Pyrazinamide (Pyrazin-AMIDE)
- Ethambutol (Etham-BUTOL)
Antituberculotic drugs:
Phase II of treatment - 4-7 months
- Rifampin (Rif-AMP-in)
- Isoniazid (ISO-niazid)
What is the definition/name for a TB that is resistant to Rifampin and Isoniazid ?
Multidrug resistant M. tuberculosis (MDR TB)
What is the definition/name for a TB that is resistant to fluoroquinolones and at least one of the second-line drugs (potentially untreatable) ?
Extensively drug-resistant M. tuberculosis (XDR TB)
What are the necessary modification in treatment needed in case of a XDR TB or MDR TB?
- Extensive use of second-line agents for an extended
period of time (18-24 months). - Surgery may be required to remove the origin of infection.
What is common for all the following drugs?
- Quinolones
- Aminoglycosides
- Polypeptides
- Linezolid
Second-line drugs for TUBERCULOSIS treatment
What is the chemoprophylactic treatment needed in case of TB exposure for HIV patients?
Isoniazid (6-9 months), Rifampin (4 months).
AIDS patients with CD4+ T-cell count less than 50 cells/μl - prophylaxis against M. avium complex: Prophylactic treatment.
Clarithromycin
Immunoprophylaxis: Vaccination with attenuated M. bovis - BCG (bacillus Calmette-Guerin for TB): Where? When? Who?
- Countries where tuberculosis is endemic
- Administered to young (less effective in adults)
- Cannot be used in immunocompromised patients (HIV infection)
Complement Fixation test (for Antibody detection)
Which bacterial antibodies are diagnosed with it?
- Mycoplasma Pneumoniae
- Chlamydophila Pneumoniae
- Chlamydia Psittaci
FITC - Definition
Fluorescein Isothiocyanate - Molecule covalently attached to Antibodies allowing for Direct and Indirect Immunofluorescence.
How does Direct Immunofluorescence work?
Checking the presence of the Antigen by confirming its attachment to FITC-labeled Antibody.
How does Indirect Immunofluorescence work?
Checking the presence of the Antigen by attachment to a Primary Antibody that will be afterwards confirmed by binding to a Second type FITC-labeled Antibody/ies.
ELISA- Definition
Give an example in Microbiology
Enzyme Linked Immunosorbent Assays
B. Anthracis detection by Anti-PA (Protective Antigen, Indirect)
T. Pallidum detection by Recombinant Antigen (Direct)
What type of Serology Test is described below?
- Premade -Antigens are affixed to the vessel Surface
- Patient serum added and Ab-Ag binding occurs
- Enzyme-Conjugated Anti-Human Antibody (E) is added
- After binding of E to Ab a substrate added is converted to Chromophore - Spectrophotometric Analysis is done.
Direct ELISA - Antibody Detection
What type of Serology Test is described below?
- Premade -Antibody are affixed to the vessel Surface
- Antigen specimen added and Ab-Ag binding occurs
- Second Premade -Enzyme-Conjugated Anti-Human Antibody (E) is added (Ab-Ag-E complex)
- After binding of E to Ag a substrate added is converted to Chromophore - Spectrophotometric Analysis is done.
Indirect ELISA - Soluble Antigen Detection
What type of Serology Test is described below?
- Proteins are separated by SDS-PAGE
- Proteins are blotted into nitrocellulose paper
- Incubation with antigen-specific antibody or patient sera (1Ab)
- Incubation with enzyme-conjugated anti human sera sera (2Ab)
- Enzyme conversion of substrate identifies the antigen.
Western Blot
What is common for all the serological test listed below?
- Real-time PCR
- Nucleic Acid Hybridization
- Immunofluorescence assays
- Indirect ELISA
- Latex Agglutination
Direct Detection of Pathogen (Antigen)
How are toxoids formed?
Acceleration of the toxins loss of pathogenicity and retainment of antigenicity
By reagents like formalin, iodine, pepsin, ascorbic acid and ketones
- 37C
- 6-9 weeks.
Diagnostic Skin tests: Tuberculin test (PPD) - with Positive and False Positive results
Purified Protein derivative of TB- Injection into skin.
Takes 48-72 hours.
Positive - red lesion greater than 15 mm indicates TB infection or previous exposure.
False Positive - Lesser reactions indicate infections with other mycobacteria or BCG vaccination passive immunity.
Diagnostic Skin tests: Tuberculin test (PPD) - with False Negative and Negative results
Purified Protein derivative of TB- Injection into skin.
False Negative -rare, due to AIDS/Steroids/Malnutrition.
Negative - 0 mm lesion should be recorded
Allergy Skin test - with Positive results
Skin Prick/Scratch/Intradermic/Patch tests all expose the skin (forearm) to the tested allergen . If - Rash, Urticaria, Anaphylaxis = Positive.
Individuals in a population will respond differently to the same dose of a toxin (Age/Sex/Weight…). How can we determine the probability of an outcome in a population, meaning the Acute toxicity of a substance?
For a population-level measure the LD50 is used!
What are Antitoxins?
Antibodies produced by toxin introduction in animals that are able to neutralize the Toxin -Collected and Given as Medical Passive immunity.
Diagnostic test using Antitoxins
ELEK’s Test - For C. Diphtheriae Diagnosis
Passive Immunization - Advantages
- Quick and Life saving
- Independent of the Immune system state
Passive Immunization - Disadvantages
- Difficult and Costly to produce
- Antibody harvesting from Humans or Animals
- Allergic Reaction occur
- Given intravenously, more complicated than vaccine.
- Short lived
Serotype
a group of closely related microorganisms distinguished by characteristic set of antigens.
Latex Agglutination- Examples for Diagnosed Microbes
Bacterial Antigens of : S. Agalactiae, S. Pneumoniae, H. Influenzae B, N. Meningitidis.
Primarily from CSF specimens.
Bacterial Non-Obligatory Vaccines
- Meningococcal vaccine: B / ACWY (Capsular Serotypes)
- Typhoid Vaccine: Live Attenuated S.Typhi
- Cholera Vaccine : Live Attenuated 01 Serotype Toxoid
- Shigella Vaccine
What are the 3 factors that determine the effectiveness of post exposure vaccines (Therapeutic) ?
(Mostly for Viral Pathogens)
- Rate of Replication of the Pathogen
- Incubation period
- Pathogenic Mechanism
Name 4 Bacteria capable of Antigenic Variation
- Neisseria Meningitidis
- Neisseria Gonorrhoeae
- Borrelia Recurrentis
- Borrelia Hermsii
Name two Bacteria causing reactive Arthritis:
Campylobacter Jejuni and Chlamydia Trachomatis
Blood culture bottles:
Importance
- Bacteriemia increases the mortality rate by 40+%
- Collecting the blood specimens efficiently is essential for diagnosis and treatment
Indications for Blood culture ( Try to remember at least 5)
- Bacteremia
- Septicemia
- Shock
- Unexplained fever of several days
- Chills and fever in patients (Infectious patients)
- Debilitating patients
- Following body piercing with signs of infection
Collection of Blood Sample:
- The venipuncture site should be __________ in order to reduce the chance of contaminant microorganisms
- Blood culture can be obtained either by using a needle and syringe or by the ______ system, consisting of a vacuum bottle and double-needle collection tube.
Collection of Blood Sample:
- The venipuncture site should be desinficiated in order to reduce the chance of contaminant microorganisms
- Blood culture can be obtained either by using a needle and syringe or by the closed system, consisting of a vacuum bottle and double-needle collection tube.
Timing of Blood Sample collection:
- Before the use of systemic _________ if possible
- Obtaining the blood culture one-half hour before a temperature ____ is ideal because the highest concentration of organisms are circulating at that time
Timing of Blood Sample collection:
- Before the use of systemic antimicrobials if possible
- Obtaining the blood culture one-half hour before a temperature spike is ideal because the highest concentration of organisms are circulating at that time
Number of Blood Sample collection:
- _ pair blood cultures (aerobic/anaerobic)
- In continuous bacteraemia: three blood cultures a day, spacing __ minutes apart
Number of Blood Sample collection:
- 3 pair blood cultures (aerobic/anaerobic)
- In continuous bacteraemia: three blood cultures a day, spacing 30 minutes apart
Volume of Blood Sample collection:
- __ ml of blood should be drawn from adults during each venipuncture.
- In infants: _ ml
Volume of Blood Sample collection:
- 20 ml of blood should be drawn from adults during each venipuncture
- In infants: 2 ml
Possible Culture medium of Blood Sample:
Culture medium of Blood Sample:
- Blood culture bottles is multipurpose and nutritionally enriched
- Commercially available blood culture media
Blood Sample Culture:
After inoculating the culture vials on the hospital floor, they are sent to the microbiology lab
clinical pathology department. Here the cultures are entered into a blood culture machine,
which keeps the samples at ____ temperature.
Blood Sample collection:
After inoculating the culture vials on the hospital floor, they are sent to the microbiology lab
clinical pathology department. Here the cultures are entered into a blood culture machine,
which keeps the samples at Body temperature.
Blood Sample Culture - Negative Results:
by monitoring carbon dioxide levels produced by
bacteria in the vials via fluorescence detected by a ___. Most cultures are monitored for _ days, after which, if the vials are negative, they are removed.
Blood Sample Culture - Results:
by monitoring carbon dioxide levels produced by
bacteria in the vials via fluorescence detected by a LED. Most cultures are monitored for 5-days, after which, if the vials are negative, they are removed.
Blood Sample Culture - Positive Results:
Microbiologist will perform a ____ Stain on the blood for a rapid, general ID of the bacteria (Initial report), The blood is later also subcultured onto agar plates to isolate which takes up to 3 days time for checking _____ & ______(C&S) process IDs the species of bacteria. Antibiotic _______ are then assessed.
Blood Sample Culture - Positive Results:
Microbiologist will perform a Gram Stain on the blood for a rapid, general ID of the bacteria (Initial report), The blood is later also subcultured onto agar plates to isolate which takes up to 3 days time for checking Culture & Sensitivity (C&S) process IDs the species of bacteria. Antibiotic Sensitivities are then assessed.
Biofilm and its Significance:
Bacteria are able to adhere to medically important surfaces such as:
Biofilm and its Significance:
Bacteria are able to adhere to medically important surfaces such as catheters, either urinary or Intravenous, artificial heart valves and other Implants.
Biofilm and its Significance:
__% of all water living bacteria live as biofilms on surfaces.
Biofilm and its Significance:
99% of all water living bacteria live as biofilms on surfaces.
Biofilm and its Significance: 3 steps of the formation process
- Primary Layer (Slime formation) - electrolytes, proteins etc.
- Colonization of Bacteria - many bacteria involved including different species.
- Breaking off of the biofilm - Dissemination to planktonic forms.
Biofilm and its Significance: Detection
-Electron Microscope on the side of a glass tube
Biofilm and its Significance: Reasons for Higher resistance to Antibiotics
- Inhibit the penetration of certain antibiotics - result in higher MIC and MBC.
- Slower multiplication
- Allow for more intense gene transfer between bacteria.
Biofilm and its Significance: Diseases involving biofilm
- Native Heart valve endocarditis
- Implantates: Artificial valves, Urinary Catheters, IUDs
- Chronic Bacterial Prostatitis
- Cystic Fibrosis Patients
- Periodontitis and Dental Plaques
Common causative agents of Meningitis in neonates
- E. coli
- S. Agalactiae
- Listeria Monocyogenes
Common causative agent of Meningitis in postnatal period
- Hemophilus Influenza B
- Listeria Monocyogenes
Common causative agents of Meningitis in Adults
- Listeria Monocyogenes
- N. Meningitidis
- S.Pneumoniae
- S. Aureus
- Leptospira Interrogans (Serous - unique)
- Mycobacterium Tuberculosis (Subacute)
Clinical Manifestations of Meningitis: 5 including Brudzinski Sign and Kernig Sign
- Headache, Fever, Nuchal Rigidity
- Neck flexion may cause flexion of the legs (Brudzinski)
- Limitations in leg extension when knee flexed (Kernig)
- Obstruction of Flow of CSF leading to Hydrocephalus
- Brain Infarcts and Neurological Deficits
Acute Meningitis - Diagnosis
CSF smear examination:
- Granulocytes Presence
- Low Glucose
- Bacteria Presence
Common causative agents of Diarrhea in neonates
- Salmonellosis
- E.Coli subspecies
- Campylobacter
- Yersiniosis
- Shigellosis
- H.Pylori
- C. Difficile; Pseudomembranous Colitis
- Aeromonas Hydrophila
- Plesiomonas Shigelloides
Diarrhea - Types and their Duration
3 non-solid stools or more per day
- Acute Diarrhea <14 days
- Persistent diarrhea >14 days
- Nosocomial Diarrhea: 72 hours after admission
Diarrhea -Biochemical Tests:
- Clinically: Bloody/Watery and Onset
- Selective Culture Media: EMB, Brilliant-green, McConkey
- Serology Methods using stool as a sample; Toxins/Bacterial Antigens
UTI - Important Typical pathogens
- E.Coli (1st most common cause)
- Streptococcus Saprophyticus (2nd Most common)
- Proteus Mirabilis (Kidney Staghorn stones)
- Klebsiella Pneumoniae
- Enterobacter Cloacae
- Serratia Marcescens
- Pseudomonas Aeruginosa
- Mycobacterium Tuberculosis
UTI - Important Atypical pathogens
- Ureaoplasam Urolyticum
- Chlamydia Trachomatis
- Mycoplasma Hominis
- Neisseria Gonorrohoeae
- Treponema Pallidum ssp
Manifestations of UTI:
- Pyuria (>5 WBCs, in microscopy) (Could be Sterile as well)
- Significant Bacteriuria: >105 CFU/ml
- Asymptomatic
- Symptomatic:Acute Urethral Syndrome
Methods of Sampling:
- Urine in sterile container
- Urine taken with catheters
- Uricult-anaerobic culture for urine sample
- Puncture of bladder
What are the causative agents for these classic STD?
- Syphilis
- Gonorrhea
- Ulcus Molle (Chancroid)
- Lymphogranuloma Venereum
- Granuloma Inguinale
- Syphilis → T.Pallidum (spp Pallidum / endemicum)
- Gonorrhea → N.Gonorrhoeae
- Ulcus Molle (Chancroid) →H.Ducreyi
- Lymphogranuloma Venereum →Chlamydia Trachomatis
- Granuloma Inguinale →Klebsiella Granulomatis
What is a predisposing bacterial factor that allows for bacterial vaginosis (STD)?
Unbalanced Vaginal Flora - mainly when Lactobacillus spp is missing or diminished and replaced by Facultative/Obligate Anaerobes!
What is the normal Anaerobe/Aerobe ratio in the Vagina? and in Bacterial Vaginosis?
Normal - 5/1
Bacterial Vaginosis - 100/1
Symptoms of Bacterial Vaginosis:
Symptoms of Bacterial Vaginosis:
- Increased vaginal discharge that often smells like fish
- Discharge is White/Gray
- Burning Sensation in urination may occur
How are STD samples collected? (Instrument)
Cytobrush
Which STD causing bacteria are not diagnosed with Serology? How are they diagnosed, Otherwise?
- N.Gonorrhoeae
- Chlamydia Trachomatis
- Mycoplasma and Ureaplasma
- Diagnosis by PCR (Called NAAT in Chlamydia)
How are Mycoplasma and Ureaplasma are cultured? (STD)
Eaton Agar
Syphilis - Screening (Aka Non-Specific):
Antigen Used
Antibody Used
Antigen Used - Cardiolipin from catale’s heart
Antibody Used - From damaged cells during early phase, present on cell surface of T.Pallidum
Syphilis - Screening (Aka Non-Specific):
Wassermann reaction
Complement fixation test:
- Negative: Lysed Sheep’s RBCs
- Positive: RBCs remain intact (Ag-Ab interaction fixate Complement)
Syphilis - Screening (Aka Non-Specific):
VDRL
Venereal Disease Research Laboratory:
-A drop of Cardiolipin is placed on a slide with a drop of serum for microscopical investigation
Diagnosis of Syphilis - Specific Methods for Diagnosis:
TPI
T. Pallidum Immobilization test:
Living T.Pallidum = Antigen, Complement of Guinea Pigs.
If in a mixture with IgGs from Serum there is no locomotion because of a Ab-Ag-Complement reaction this is a Positive result.
Diagnosis of Syphilis - Specific Methods for Diagnosis:
TPHA
T.Pallidum Haemagglutination test: (Flocculation)
-Aggregation of T. Pallidum sensitized (with Ab) RBCs with exposure to patient serum means Positive result.
Diagnosis of Syphilis - Specific Methods for Diagnosis:
FTA-ABS
Fluorescent Treponemal Antibodies:
- Antigen = Killed T.Pallidum , Second Antibody = Fluorescein labeled Anti-Human Antibodies, First Antibody from Patient Serum!
- Positive results: Bright yellow fluorescence upon triple particle Immunocomplex formation.
A human first becomes colonized by a normal flora at the moment of:
A human first becomes colonized by a normal flora at the moment of birth and passage through the
birth canal.
Exposure of an individual to an organism can lead to one of three outcomes:
- Transient colonization (hours to days)
- Permanent colonization
- Cause disease
Beneficial effects of Normal Flora:
The normal flora synthesizes and secretes _______ in excess of their own needs, which can be
absorbed as nutrients by their host. For example, in humans, enteric bacteria secrete _______ and
_________ , and lactic acid bacteria produce certain ________ .
Beneficial effects of Normal Flora:
The normal flora synthesizes and secretes vitamins in excess of their own needs, which can be
absorbed as nutrients by their host. For example, in humans, enteric bacteria secrete Vitamin K and
Vitamin B12, and lactic acid bacteria produce certain B-vitamins
Beneficial effects of Normal Flora:
The normal flora prevents colonization of ______ by competing for attachment sites or for
essential nutrients.
Beneficial effects of Normal Flora:
The normal flora prevents colonization of pathogens by competing for attachment sites or for
essential nutrients.
Beneficial effects of Normal Flora:
The normal flora may antagonize other bacteria through the production of substances which inhibit
or kill ___-______ _____ .
Beneficial effects of Normal Flora:
The normal flora may antagonize other bacteria through the production of substances which inhibit
or kill non-indigenous species.
Beneficial effects of Normal Flora:
The intestinal bacteria produce a variety of substances ranging from relatively nonspecific fatty acids and peroxides to highly specific bacteriocins, which :
Beneficial effects of Normal Flora:
The intestinal bacteria produce a variety of substances ranging from relatively nonspecific fatty acids and peroxides to highly specific bacteriocins, which inhibit or kill other bacteria.
Beneficial effects of Normal Flora:
The normal flora stimulate the ________ of certain tissues, i.e., the cecum and certain lymphatic tissues (Peyer’s patches) in the GI tract.
Beneficial effects of Normal Flora:
The normal flora stimulate the development of certain tissues, i.e., the cecum and certain lymphatic tissues (Peyer’s patches) in the GI tract.
Beneficial effects of Normal Flora:
Low levels of antibodies produced against components of the normal flora are known to cross react with:
Beneficial effects of Normal Flora:
Low levels of antibodies produced against components of the normal flora are known to cross react with certain related pathogens, and thereby prevent infection or invasion.
Examples for Normal flora of the Skin:
- Nares, Face and Hands: Staph. Aureus
- Upper parts of Hair Follicles: Staph. Epidermidis and Propionibacterium spp.
Examples for Normal flora of the Respiratory Tract:
- Nares: Staph. Epidermidis, Staph. Aureus(20% of pop)
- Pharynx: Strep.Pneumoniae, Pyogenes, H.Influenzae, N. Meningitidis.
- Facial Sinuses and Lower respiratory tract should be sterile in healthy conditions.
Examples for Normal flora of the Urogenital Tract:
Examples for Normal flora of the Oral Cavity:
-Lactobacilli, Bacteroides, Spirochetes , Streptococcus Salivarius (First kind in neonates), Viridans Group (After teeth erupt)
Examples for Normal flora of the Gastrointestinal tract:
- Esophagus: Lactobacillus.
- Proximal Small Intestine: Lactobacilli, Bacteroides and Enterococcus Faecalis
- Distal Small Intestine: Lactobacilli, Enterococcus Faecalis with E.Coli
- Colon: More and More Coliforms, Bacteroides and Lactobacilli
Examples for Normal flora of the Gastrointestinal tract in Infants:
- Bifidobacteria is prominent in Breast Fed
- Bacteroides, Enterococci are prominent in Bottle fed
Transient Bacteremia
Self Limited, due to multiplication of infected tissue (Abscess)/Trauma to Mucosal Surface/Localized infection
Intermittent Bacteremia
Microorganisms from an infected site are spasmodically released into the blood. may be from extravascular abscess, empyemic cavities or diffuse infections.
Continuous Bacteremia
Severe, Overwhelms the host defences. Intravascular site of infection: Endocarditis/ Catheters/ AV fistulas/ Long-term Cannulae.
Sepsis
Excessive Inflammation caused by a combination of factors related to the particular blood invading pathogen and the status of the immune system. May lead to Shock and Multi-organ failure.
SIRS (Systemic inflammatory response syndrome) - diagnosed by two or more of signs,
due to pancreatitis, burns, trauma, other. What are the signs? (Try to remember 3)
Ø Temperature > 38° or < 36°
Ø Tachycardia > 90 BPM
Ø Respiratory rate > 20 breaths / min or PaCO2 < 4.3 kPa
Ø WBC count > 12 * 109 or < 4 * 109 or > 10% immature forms
Steps of Sepsis: by severity
1) SIRS - According to Parameters
2) Sepsis - SIRS with a suspected/Confirmed infection
3) Severe Sepsis - Hypotension, Signs of End Organ Failure and Elevated Lactic Acid in Blood.
4) Septic Shock - Persistent symptoms of Severe Sepsis
Nosocomial infection - Definition
Hospital-acquired infections (vs. community-acquired). Includes any infections acquired in the hospital
during 48 hr from Staff or Equipment.
Cause of Nosocomial infections:
When health care workers do not practice proper hygiene. Medical procedures that bypass the body’s natural protective barriers increase the risk of
nosocomial infections
What is the most common route of nosocomial infections?
Direct contact
Common Microbes responsible for Nosocomial Infections: (Remember at least 5)
- Ventilator-associated/Hospital-acquired pneumonia
- Staphylococcus aureus
- MRSA
- Pseudomonas aeruginosa
- Acinetobacter baumannii
- Clostridium difficile
- Urinary tract infections
- KES
- Enterococci
Iatrogenic infections - Definition
Infections transmitted during medical treatment and care. Any effect on a person, due to the effect of
healthcare professionals, that does not support a goal of the person affected.
Iatrogenic infections - Causes
- Side effects of possible drug interactions, overuse of drugs, adverse effects of drugs
- Complications following a surgical procedure
- Medical error, including wrong prescriptions / ineligible writing
- Negligence
