Bacteriology Notes

 Bacteria Eubacteria Unicellular, prokaryotic Microbiology Overview Eukarya Protis...

 Bacteria Eubacteria Unicellular, prokaryotic Microbiology Overview Eukarya Protista Unicellular Microbiology is the study of and microorganisms, a large and diverse group multicellular of microscopic organisms that exist as Fungi Multicellular, single cells or cell clusters. eukaryotic This study aims to know the causative Plantae Multicellular, agents of infectious diseases along with its eukaryotic environmental importance and industrial Animalia Multicellular, importance eukaryotic Scope Of Microbiology Classification Of Cells Medical microbiology Prokaryotes Characteristics Eukaryotes Public health and epidemiology DNA within a No Yes Immunology nuclear membrane Industrial microbiology Binary fission Mitotic division Mitosis Agricultural microbiology DNA associated with No Yes Environmental microbiology histone More than One (1) Chromosome number one Microorganism Membrane bound A minute organism and cannot be viewed No Yes organelles by the unaided or naked eye. 70s Size of ribosomes 80s Cell wall containing Taxonomy Yes No peptidoglycan Means to classify, name and identify organism. Comparison Between Different Classification System Microorganisms Genus Contains similar species Protozoa Family Contains similar genera Viruses Bacteria Fungi And Order Contains similar families Helminths Class Contains similar orders Cells No Yes Yes Yes Phylum Contains similar classes Diameter 0.02 – 3-10 um 15-25 um 1-5 um Contains similar divisions (um) 0.2 um (yeast) (trophozoites) Kingdom either Nucleic DNA both DNA both DNA both DNA Nomenclature acid or and RNA and RNA and RNA Naming of organism according to RNA established rules and guidelines set by the Type of None Prokaryotic Eukaryotic Eukaryotic international Code of Nomenclature of nucleus Bacteria Identification Two General Categories Bacterial Shape and Size Phenotypic Shape Name Genotypic Cocci Overview Domain Kingdom Sphere Archaea Archaebacteria Unicellular, prokaryotic (do not cause disease) K.K. ONG LO | 1  Protects against mechanical disruption of the cell Essential for survival of bacteria Bacilli Rod Note Christian Gram – developed Gram stain Spiral Spirochetes Different or Pleomorphic variable shapes Bacterial Arrangement Arrangement Name Dyes Used for Gram Staining Crystal violet or Methylene Primary Stain blue Counter stain Safranin 2 pair Diplococci Decolorizer acetone and alcohol (95%) Components of The Backbone of Peptidoglycan N- acetylglucosamine (NAG) N- acetylmuramic acid (NAC) Chain Streptococci o Tetrapeptide side chain o Cross bridge Cluster Staphylococci Bacterial Cell Wall Peptidoglycan – also called as o Murein o Mucopeptide Function BETA 1-4- links NAG &AC Gives the bacterial cell shape and strength Tetrapeptide side chains are attached to N- to withstand changes in environmental acetylmuramic acid (NAC) osmotic pressures K.K. ONG LO | 2  Alanine Wall Teichoic Acid D-iso glutamine Attached to the peptidoglycan. Lysine D—Alanine Lipoteichoic Acid Attached to the membrane Pentaglycine/ Pentapeptide – connects two tetrapeptide side chain which is present only in gram-positive bacteria K.K. ONG LO | 3  amino acids, and amino sugars Present within the murein layer; Mycolic makes the cells None Acids more refractory to toxic substance Stains Not immediately decolorized Gram Color of the primary stain is Positive seen PURPLE Color of the counter/ Gram secondary stain Negative Easily decolorized PINKISH REDISH Liposaccharides (LPS) Gram-negative bacterial cell wall has Essential Components of Bacteria liposaccharide (LPS) called endotoxin. Cell Wall Characteristics Function Site of oxidation Cytoplasmic Lipoprotein Components of Liposaccharides (LPS) and transport of membrane without sterol Lipid A enzyme RNA and Protein Toxicity of gram-negative bacteria. Ribosomes in 50s and 30s Protein synthesis subunits O Antigen Nucleoid DNA Genetic material Immunogenic (stimulate body to produce Participates in antibody) Invagination of Mesosome cell division and plasma membrane secretion Porin Contains Porin is one of the compositions of gram- Space between hydrolytic negative bacteria where there is passive plasma membrane Periplasm enzymes, diffusion for sugar, amino acid, and and outer including beta certain ions. membrane lactamase Non-Essential Components of Bacteria Components Characteristics Function Gram- Protect against Gram -Positive Capsule Polysaccharide Negative phagocytosis Peptidoglycan Pilus or Attachment, Thicker Thin Glycoprotein Layer Fimbriae conjugation Present Mediates Absent Pentapeptide Attached in the Glycocalyx Polysaccharides adherence to linked directly tetrapeptide surfaces glycerol or ribitol Flagellum Protein Motility phosphate Resistance to Teichoic Acid polymers None Keratin like coat, Spore heat and combined with dipicolinic acid chemicals various sugars, Glycogen, lipids, Site of nutrients Granule polyphosphates in cytoplasm K.K. ONG LO | 4  Note Flagellar Arrangement Plasmid The bacteria can be classified based on its o Aside for pilus, it carries genetic flagella. bacteria thru conjugation; Arrangement Characteristic therefore, the resistance also One flagellum transfers Biofilms o If bacteria can make a biofilm, like Monotrichous a community, they are hard to treat. Biochemical Test o Determines motility of bacteria; Two flagella 24-48 hrs. if there is growth away in stab area which indicates motile. Hanging Drop Method o Involves slide with depression, Amphitrichous NSS, sample Spores o Doesn’t die with boiling ▪ Autoclave – for sterilization also, has PI 2 or more flagella steam under pressure = 120 degree C Lophotrichous Flagella For motility Flagellin Composed of H antigen and considered as More flagella highly antigenic. that encircle the Considered as the major protein of flagella bacterial cell and principal component of bacterial flagella. Peritrichous Pili (Fimbriae) Provides attachment and for conjugation. Shorter and finer than the flagella K.K. ONG LO | 5  Pilin Produced by some bacteria in response to Major protein of pili nutrient limitation Resistance to heat and chemicals Adhesive Resistance of spore is due to dehydrated Minor protein of pili state, calcium dipicolinate. Formed by gram positive rods: bacillus and Note: clostridium spp. F (+)– donor cell, genetic material of the bacteria Plasmid – carries the genetic information F (-) – has no plasmid Process Pili or pilus conjugates until they are joined, once the F (+) and F (-) are fully joined, the plasmid is transferred to the F (-) After it is transferred, they will separate, and the F (-) will become the F (+) Significance: Its significance is for transferring of genetic material to another bacteria is associated with antibiotic resistance. Sporulation Formation in filaments, unfolding which results to double membrane then engulf. There will be a synthesis of spore wall and cortex. There will be coat deposition. This will now mature and go out to cell lysis. Spores Allows the bacteria to thrive in a harsh environment. Resistant in heat and chemicals. small dehydrated, metabolically quiescent forms (reversible stage of non-division of cell) K.K. ONG LO | 6  Moderate potential hazard to personnel and environment; limit the Universal Precaution access of personnel treating all body fluids as infectious. Has known cure causes mild disease and difficult to Biosafety BSL 2 contract via aerosol in a lab setting. application of the combination of laboratory Engineering control: sink, fume hood, practices, procedures and safety equipment to shower prevent release of potentially infectious Ex. Chlamydiae, Hepatitis A, B, and disease or organism in laboratory as well as C, Influenza A, Lyme Disease, the community. Salmonella, Mumps, and Measles. measures employed when handling microbes can be either indigenous or biohazardous materials to avoid infecting the exotic; causes serious or potentially specimen, laboratory personnel, community lethal disease through respiratory (environment itself) transmission Prevent: control measures: BSL 3 o laboratory acquired infections o PPE which includes mask w/ o secondary infections in general respirator. population Ex. HIV, H1N1 Flu, Yersinia pestis o environmental/animal contamination (plaque), TB, SARS, Rabies, West Nile Virus, Ricketts * Elizabeth Griffin – a researcher died from a no known cure or vaccines. drop of virus from a monkey same controls + highly secured organisms TOP 4 Accidents Resulting Infection dangerous and exotic, posing high BSL 4 1. Spillages and splashes risk of aerosol-transmitted infections 2. Needle and syringe by microbes which are frequently 3. Sharps objects, broken glass fatal. 4. Bite or scratch from animals Ex. Ebola, Small pox CDC Biosafety Levels Control Measures organisms – determines the risk and hazard Engineering controls all controls are anchored to biosafety levels Administrative controls which is also based on the risk and the hazard Practices and procedures involved. Personal Protective equipment (PPE) → all are dependent to BSL Biological safety cabinets Engineering (BSC) Controls Enclosed containers Animal Insulators Training o Lab safety o Toxin-specific session o Annual refresher with not known to cause disease Administrative Occupational minimal potential hazard to Controls Physician laboratory personnel & Medical Surveillance environment. o Vaccine and antitoxin BSL 1 Engineering control: sink, fume hood available Ex. Canine hepatitis, Non- Standard Control access to the pathogenic E. coli, Non-infectious Microbiological laboratory bacteria. Practices K.K. ONG LO | 7  Wash hands after working Basic Principle of Biosafety Cabinet: & before leaving the lab Personnel protection is provided through a safety handle of sharps continuous stream of inward air known as perform all procedures to inflow, which helps prevent aerosols from minimize the creation of escaping through the front opening. splashes and/or aerosols The exhaust air, which is exhausted into the surrounding containment zone is HEPA Types of Cabinets filtered to protect the environment. Fume hood removes toxic chemicals HEPA & ULPA Filter No HEPA filter HEPA High efficiency particulate air not for biohazard agents o 99.99% at 0.3 microns for obnoxious reagents ULPA Ultra-low penetration air o 99.99% at 0.12 microns Laminar Flow Chart product protection Proper Use of the Biosafety no personnel protection Cabinet not for biohazard agents or chemical fumes 1. Disinfect the interior surfaces with a flow of air can go outside the environment. disinfectant effective against the infectious no filter; contamination can is possible. materials and toxins used in the laboratory, allowing an appropriate contact time Biosafety Cabinets (BSC) 2. Assemble all materials required for manipulation and load into the BSC Class I Personnel and environment 3. Care should be taken not to overcrowd or protection block the front grilles rear grilles to prevent the appropriate airflow from being comprised Personnel, product and 4. After loading materials in the BSC, allow environment protection sufficient time for the airflow to stabilize negative pressure ventilated before initiating work Class II cabinet 5. This will be specified in the manufacturer’s provide HEPA filtered, instructions and is generally 3-5 minutes recirculated airflow within the 6. Perform operations as fat to the rear of the cabinet work area as reasonable Exhaust air is HEPA filtered 7. Ensure that elbows and arms do not rest on Provides personnel and product the grille or work surface protection 8. Allow excessive movement of hands and HEPA filtered air is discharge arms through the front opening it will disrupt Class II A into the room the airflow and allow contaminants to enter 70% exhaust, 30 re-circulate or escape the BSC typical uses: bacterial, viral, 9. Suspend open flames are prohibited fungal, parasitic 10. Non flame alternative should be use like Class II B HEPA filtered air is discharge out micro incinerator or sterile disposable the room inoculation loop 2 classes: 11. Upon completion of work decontaminate Class II B1 70% exhaust, 30 re-circulate 12. Use UV light 100% exhaust Materials Class II B2 Uses: bacterial, viral, fungal, conical base with a parasitic, prions, cytotoxic cylindrical neck. used for pH titrations Class III Personnel and environment preparation of microbial protection cultures Erlenmeyer Flask → HEPA filters not for chemical vapors K.K. ONG LO | 8  as the steam goes up, Inoculated Loop – pressure will be lowered inoculate specimen on the = STEAM UNDER petri dish PRESSURE Inoculated Needle - use for QC: may spore, dapat stabbing (SIM Test) mapatay niya yung Inoculating spore para masabi na Loop/ Needle working ang autoclave amount of media suspended depends on the size of petri dish Synthetic Culture Media Recommended: ½ usual media in powdered form Petri Dish always in 1,000 Ml or 1 L measure the synthetic culture media (powdered) Top Load Containment Balancer safe methods, facilities and equipment for heating of culture media managing infectious materials in the (near boiling, presence laboratory environment where they are being of bubbles at the bottom handled or maintained. of Erlenmeyer) Purpose: reduce or eliminate exposure of laboratory workers, other persons, and the Hot Plate outside environment to potentially hazardous agents. Three Main Elements of Safe Containment for Microorganisms: Good laboratory practices and techniques Test Tube And Safety equipment Test Tube Rack Facility design storage of inoculated specimen Good Laboratory Practices and Temp – 37 centigrade; stored for 24-48 hours Techniques except for M. Wash your hands with soap and water before Incubator tuberculosis – 7 days and after entering the laboratory. (1) to slow down the growth Absolutely no food, drinks, chewing gum or of bacteria; using inoculated smoking is allowed in the laboratory. Do not Refrigerator petridish put anything in your mouth (pencils, pens, or (2) freshly prepared culture labels). media Only closed - toe shoes are to be worn in the all of the materials must laboratory (sandals are not permitted). be autoclaved Keep hands and other objects away from your face, nose, eyes, ears and mouth. The Once its-on = heating application of cosmetics in the laboratory is Gauge will increase to prohibited. 100 degrees Work areas and surfaces must be disinfected Autoclave before and after use. Once it reached 120 degrees, heating will Laboratory coats must be worn and buttoned stop → Sterilization for when performing any exercise or procedure 15 minutes in the laboratory. Laboratory coats should If opening, sa gilid not be worn outside the laboratory. ioopen to prevent burn K.K. ONG LO | 9  Protective eyewear must be worn when D. Face Shield or Goggles performing any exercise or procedure in the Wash hand laboratory. Gloves should be worn at all times. Long hair should be secured. Standard Microbiological Safety All unnecessary books, purses, briefcases, and gadgets must be kept off the working Practices areas. A. Disinfectants Never pipette anything by mouth (including Microorganisms have different water). Always use pipetting device. susceptibilities to various disinfectants. As a Label all materials with your name or group surface disinfectant, 70 % alcohol is generally number, date, section and any other effective. applicable information (e.g media, organism An effective general disinfectant is a 1:100 etc.). (1%) dilution of household bleach (Sodium Dispose of wastes in their proper containers hypochlorite) in water; at this dilution you (take note of sharps and biohazard waste). can use this bleach to wipe surfaces of When handling chemicals, note the hazard benches, hoods and other equipment. code on the bottle and take the appropriate If bleach is used; wipe down the area with 70 % precautions indicated. alcohol to inactivate the bleach Do not pour chemicals down the sink. If Sodium hypochlorite is used as a Report any broken glass, especially those disinfectant, the standard 1% dilutions should containing infectious materials. be made daily from a stock solution. If you are injured while working in the laboratory, immediately report it to your B. Sharps laboratory instructor. Dispose sharps in designated puncture - proof, Any chemical or biological fluid spills must leak - proof and sealable sharps containers. be immediately reported to your laboratory To minimize incidence of finger prick, do not instructor for proper cleaning and bend, shear, break, recap, remove from decontamination procedure. disposable syringes or manipulate by hand Always wipe and clean the lenses of your used disposable needles microscope before and after use. Non- disposable sharps should be placed in a Always follow the Universal Precaution. labeled discard pan for decontamination Do not remove any materials from the before cleaning laboratory without the written permission of Broken glassware should be removed by the laboratory instructor. mechanical means (brush and dustpan, tongs or forceps) and should not be handled by Sequence For Doffing PPE hand. A. Outermost Layer of Gloves: C. Aerosols Remove gloves by grasping the outside of the Centrifugation, vertexing and vigorous glove near the wrist carefully peeling it off and mixing should be performed in closed turning it inside out. containers. With the ungloved hand, carefully slide a Loops containing infectious material should finger under the wrist of the glove to peel it off be dried in hot air above a burner before from the other hand turning it inside out. flaming. Wash hand after removing the gloves Cool inoculating wires and loops after flame sterilization by holding them still for 5 to 10 B. Mask seconds. Wash hand after removing the mask D. Management of Spills C. Gown Stop work immediately and alert others of Peel the gown away from the neck and spill. shoulders turning the gown inside out to keep Assemble clean -up materials (disinfectant, the contaminated portion away from the paper towels, biohazard bags and forceps). body, rolling it into a bundle. Wash hand after removing the gown K.K. ONG LO | 10  Put on appropriate PPE including laboratory coat, shoe covers, gloves and eye/face protection. Three separate but interrelated areas of Initiate cleanup with disinfectant as follows. bacterial taxonomy a) Place paper towels or other absorbent Classification: categorization of material over spill area. organization into the taxonomic group b) Carefully pour disinfectant concentrically International committee on systematic beginning around the edges of the spill prokaryotes – the committee that names and then working towards the center onto the paper towels. Avoid splashing or the bacteria generating aerosols droplets. Nomenclature: naming organism by c) Allow disinfectant to remain in contact international rules with spill for at least 20 minutes. Identification: practical use of d) Apply more paper towels and wipe up by classification scheme starting outside of the spill working towards the center. e) Clean spill area with fresh towels soaked Taxonomic rank in disinfectant. Linnaean taxonomy: uses formal f) Dispose of all towels or absorbent taxonomic ranks materials using appropriate biohazardous Kingdom, phylum, class, order, family, waste disposal procedures. If any sharp genus specie objects are present, use forceps, a dustpan or stiff cardboard and discard in a sharp’s container. Nomenclature g) Remove protective clothing and segregate Binomial system for disposal or cleaning. First name genus h) Wash hands. o ex. Staphylococci Second name o Staphylococci epidermidis Both genus and specie name are italicized An increase in bacterial numbers Does not refer to an increase in size of the individual cells Binary Fission Bacterial doubling time Escherichia coli – 20 mins Mycobacterium tuberculosis – 18 hours Mycobacterium leprae – 14 days K.K. ONG LO | 11 Generation time is the time required for a cell to Metabolic activity is active and is most divide and its population to double. The preferrable for industrial purposes generation time among organisms vary according Sensitive to adverse conditions to environmental conditions such as temperature or pH level. Most bacteria have a generation time Stationary phase of 1-3 hours while other species can require up to Period of equilibrium 24 hours per generation Metabolic activity of surviving cells slows down which stabilizes the population Measurement of bacterial growth Cause of discontinuity of exponential A. Determination of bacterial mass growth is not always clear 1. Getting the dry weight May play a role in the exhaustion of 2. Measurement of optical density in nutrients, accumulation of waste spectrophotometry products and harmful changes in the pH 3. Turbidimetric and nephelometer 4. Nitrogen determination Death phase 5. Measurement of cell volume after Logarithmic Decline phase centrifugation Provides little to no nutrients hence, the B. Determination of cell number bacteria die 1. Stained slide method Continues until a small fraction of the 2. Counting chamber (Petroff-Hauser population is diminished Counter) Some population dies out completely 3. Coulter counter 4. Calibrated wire loop 5. Pipet dilution method 6. Pour plate method Physical Requirement Temperature Bacterial Growth Curve pH Modeled in 4 different phases Osmotic pressure Temperature Psychrophiles Polyunsaturated fatty acids in their cell membranes which gives fluid nature to the cell membrane even at a lower temperature Bacteria that can grow at 0C or below but the optimum temperature of growth is 15C or below and maximum temperature is 20C Lag phase Bacterial adaptation Facultative Psychrophiles Period of little or no cell division Those that can grow even at 0C but Can last for 1 hour or several days optimum temperature of growth is 20- Cells are NOT dormant 30C Undergoing a period of intense metabolic activity: DNA and enzyme synthesis Mesophiles Log phase or exponential phase Grow best between 25-40C Also known as logarithmic increase Optimum temperature for growth is 37C Rapid multiplication Most human pathogens are mesophilic in Cellular respiration is most active during nature this period Ex: E. coli, Salmonella, Klebsiella, Staphylococci K.K. ONG LO | 12  Thermophiles Osmotic Pressure Grow best above 45C Osmophilic Contains saturated fatty acids in their cell Requiring high osmotic pressure membrane so their cell membrane does not become too fluid even at higher Halophiles temperature Moderate to large salt concentrations Ex: Streptococcus thermophiles, Bacillus Cell membrane is made up of stearothhermophilus, Thermus aquaticus glycoprotein with a high content of negatively charged glutamic acid and Facultative thermophiles aspartic acid. So high concentrations of Thermophiles capable of growing in Na+ ion concentration is required to mesophilic range shield the negative charge Ocean water contains 3.5% salt, most Obligate thermophiles bacteria are present in the ocean True thermophiles are called as Ex: Archeobacteria, Halobacterium, Stenothermophiles Halococcus Hyperthermophiles Extreme or Obligate Halophiles Optimum temperature growth of above Require a very high salt concentration 80C (20-30%) Mostly Archeobacteria Bacteria in the dead sea, brine vats Monolayer cell membrane of Archeobacteria is more resistant to heat Facultative Halophiles and they adapt to grow in higher Do not require high salt concentration for temperature growth but can tolerate up to 2% salt or Ex: Thermodesulfobacterium, Aquifex, more Pyrolobus fumari, Thermotoga Chemical Requirement pH Carbon Acidophiles Nitrogen, Sulfur and Phosphorus Grow best at an acidic pH Trace Elements The cytoplasm is acidic in nature Oxygen – important for classification of Some acidophiles are thermophilic in bacterial group nature, called Thermoacidophiles Organic Growth Factors Ex: Thiobacillus thioxidans, Thiobacillus ferroxidans, Thermoplasma, Sulfolobus Carbon Structural backbone of living matter Alkaliphiles Grow best at an alkaline pH Chemoheterotrophs Ex: Vibrio cholerae optimum pH of Get the most of their carbon from the source of growth is 8.2 their energy, organic matter (protein, carbohydrates and lipids) Neutralophiles Grow best at a neutral pH (6.5-7.5) Chemoautotrops and Phototrophs Most of bacteria grow at a neutral pH Derive their carbon form carbon dioxide Ex: E. coli K.K. ONG LO | 13  Nitrogen, Sulfur and Phosphorous Growth Factor For the synthesis of protein synthesis Purines and pyrimidines – synthesis of Nitrogen and sulfur – protein synthesis nucleic acid Nitrogen and phosphorous – synthesis of Amino acids – synthesis of proteins DNA, RNA and ATP Vitamins – needed as coenzyme Trace Elements Essential for certain functions of certain enzymes processes of transfer of genetic information Assumed to be naturally present in tap Mutation – causes the bacteria to gain new water and other components of media characters or develop virulence in bacteria Oxygen Bacteria contains sickle chromosome with circular DNA in nucleoid Prokaryotes have a simpler cell structure with Obligate aerobes DNA located in the nucleoid Most growth is at the upper portion Organism thrives at the present of oxygen Plasmid Obligated to the supply oxygen, they will Plasmid can be integrated with die without oxygen chromosomal DNA Plasmid is capable of replicating Obligated anaerobes independent chromosomes, can act as a Growth is at the bottom vector in genetic engineering Organism thrives without oxygen Present if gram (+) and gram (-) Organisms should not have any oxygen May encode genetic information for or else they will die properties: Ex: Clostridium bacteria that causes o Resistance to antibiotic tetanus and botulism o Bacteriocin production – toxic protein, lethal for other bacteria Facultative anaerobes o Enterotoxin, exotoxin Scattered growth, more on the top o Enhanced pathogenicity Thrives on the presence or absence of o Reduced sensitivity to mutagen oxygen o Degrade complex organic Complex set of enzymes molecule (bio mediation) Two types: Aerotolerant anaerobes Transmissible Growth is very scattered Cell to cell transfer by conjugation Cannot use oxygen but can tolerate small Large and contains a dozen genes for the amounts of oxygen synthesis of sex pilus, used for conjugation and enzymes required for Microaerophiles transfer Require oxygen, but at low concentrations. Sensitive to toxic forms of oxygen. K.K. ONG LO | 14  Non-transmissible Altered nucleotide sequence expresses Small, do not contain the transfer gene new or altered characteristic present in many copies per cell Ex: Virulent to non-virulent Acquired antibiotic resistance grows in Episomes the body without inhibition Integrated form of plasmid with DNA Become a prominent organism First Diagram Phenotypic variation occurs when genes changes in response to the environment o Binary fission produces two but reversible daughter cells o There are some cases where it can Second Diagram be irreversible especially during o Plasmid can be integrated in some the cell cycle where the damage bacteria DNA cannot be fix. o If the bacteria undergo binary o In cell cycle, it has control fission, it will also produce mechanism, when there is integrated plasmid called damage DNA, the cell cycle will Episomes stop and it will try to fix the DNA. But if it cannot fix the DNA, it will undergo apoptosis and the cell cycle will not continue. o As humans, we have protooncogenes. If there are mutation or carcinogenic substances, this will become oncogenes and it will affect p53 which is important in the cell cycle control. Hence, there will be no fixing of DNA, but it will still proliferate. Genome sum total of gene that make up the genetic apparatus of cell established as genotype Phenotype is the physical expression in an environment and can be reversible Mutation can occur in any sequence, inevitable, useful for survival Principles of Genotypic Variation All genes are susceptible for mutations Mutations but all mutations are not expressed genotypic by transfer of genes Lethal mutation is harmful and destroy o Transformation the vital functions o Transduction o Conjugation Transduction transfer of a portion of DNA from one Mutation in Bacteria bacteria to another by Bacteriophages. Bacteria multiply by asexual binary fission K.K. ONG LO | 15  Bacteriophage replication, budding and the cell will lyse are bacterial virus that infects bacteria. in order to release phage virions and The infected bacteria will now acquire a other viruses. new character. This will now become a bacteriophage can infect only certain bacteria bearing a which is ready to infect other cells. receptor to which they can bind. Hence, Bacteriophage doesn’t just infect any they need a particular receptor. other bacteria because it has specific It cannot cause human disease. This is receptor where the tail will bind. not harmful in humans but it can transfer its DNA on its infected bacteria. Lysogenic Cycle The link between bacteriophage to actual In Lysogenic Cycle, there is integration of human pathogen is their ability to alter the phage DNA in the host DNA where the genome of non-virulent bacteria this is called as prophage due to its strain thus producing more virulent combination of the phage DNA and the strain. bacterial chromosome. Human pathogens can be infected by After it has been integrated in the bacterial bacteriophage where this might be chromosome, it will undergo binary coding for virulence. Therefore, non- fission where it will produce 2 daughter virulent bacterial strain can now become cells with the same genetic material as the virulent. mother cells. Head – contains DNA o Ex. If there is integrated phage that is Tubes – passageway od DNA virulent and codes for resistance. Tail – for attachment Therefore, the daughter cells will also have this new characteristic and become virulent as well. Lysogenic bacterium means there is integration already of phage and host DNA. They will reproduce normally by binary fission. Occasionally, the prophage can be removed in bacterial chromosome, then there will be another combination and it will initiate another lytic cycle. 2 Cycles of Infecting Bacteria in Bacteriophage Lytic Cycle Lysogenic Cycle Process: Bacteriophage will attach to the host cells which is the bacteria using its tail In the tube portion of bacteriophage, the DNA will be released. Then, the phage DNA will become circular and it will either enter the Lytic cycle or the Lysogenic cycle. Lytic Cycle In the Lytic Cycle, a new bacteriophage DNA and proteins are being synthesized. Then, it will now be assembled into virions or bacteriophage. It will undergo K.K. ONG LO | 16  Conjugation The mobile plasmid will have a nick and Plasmid which is separated from the a single strand of DNA will now be chromosome, its genetic material will transferred to recipient cells. fuse with other bacteria for it to transfer. When both cells are synthesized, they Bacterial conjugation is beneficial in will have a complementary strand to bacteria because it can acquire a gene produce a double stranded circular that can confers survival or a new or plasmid and they will also reproduce pili novel characteristics for it to thrive in in order for both of the cells to be a viable harmful conditions. This can also utilize donor. a new metabolite. When the plasmid is transferred to the This process takes place in resistance to recipient, the end result of conjugation is antibiotics which can be transferred from having two donor which is the old donor one bacteria to another. and new donor. This new donor now can donate to F (-) Basic Plasmid because it became F (+). aka F plasmid or F (-) factor. o This can be episome which is a plasmid that is integrated in bacterial chromosome. o There is only 1 copy of F plasmid in a given bacterium, its either free or integrated. F positive or F (+) = donor o the bacteria that possess a copy of plasmid. F negative or F (-) = receiver o cells that lock F plasmid. They can function as recipient cells. Process: Transformation There is a transfer of genetic information through the activity of DNA. In this image, a gene from bacterial cell 1 is moved from bacterial cell 1 to bacterial cell 2. This process of bacterial cell 2 taking up new genetic material is called transformation. Donor cells produces pilus where this will attach to the recipient cells and it will bring the two cells together. K.K. ONG LO | 17  Staphylococcus aureus S t a p h y l o c o c c i Non-motile, gram positive, grapelike cluster Non encapsulated except a few strains produce slime or capsule Aerobic or facultatively anaerobic Catalase positive, found in soil and skins of humans Ferments carbohydrates Natural habitat: skin and nostril Produce pigments from white to deep Most virulent among the Staph species yellow Transmission: Some are normal flora of the skin, and o Direct contact mucous membranes. o Fomites There are at least 40 species belonging to o Contaminated food the Genus Staphylococci only three are of Shape Cocci clinical importance. Arrangement Cluster Family: Micrococcaceae Color Purple Genus: Staphylococcus Specie: Size 0.5 – 1.5 um o Staph. aureus o Staph. epidermidis Structure of Staph. aureus o Staph. saprophyticus Morphology And Identification Spherical cells about 1 micrometer in diameter, arranged in irregular clusters. Single pairs tetrads and chains are also seen Non motile and do not form spores. Gram positive but on aging culture many cells become gram negative. Micrococcus species resemble Staphylococci, they are rarely associated with disease. A- toxin and PBL – destroys the WBC Culture Clumping factor – helps the S. aureus hide from the immune system Grow on most bacteriologic media under Penicillin binding-protein – render the aerobic or microaerophilic conditions. resistance to penicillin Grow best at 37C Form pigment best at room temperature (20-25C). the characteristic growth on media are Cell wall Associated Structure round, smooth, raised and glistening. Capsule Species Formation On Culture Media o Adhere to host cell S. aureus Forms gray to deep golden o Resist phagocytosis yellow colonies. Peptidoglycan S. epidermidis Usually gray to white on o Inhibits inflammatory response primary isolation. Protein A S. saprophyticus o Binds to Fc region of IgG, exerting antiphagocytic effect K.K. ONG LO | 18  Toxic shock syndrome toxin (TSST) o Produce fever, skin rashes, diarrhea, death to shock Exofoliatin toxin o Breaks intercellular bridges in the stratum granulosum of the epidermis and causes separation from the underlying tissue resulting in a blistering and Clotting factor (coagulase) exfoliating disease of the skin o Cause organisms to clump in the presence of plasma Extracellular Enzymes Free coagulase Note: o Clots plasma IgG is an antibody, which has heavy chain Staphylokinase (blue in the picture) and light chain (red in o Degrades fibrin clot the picture). Both edges of the chain has Hyaluronidase antigen binding sites where it binds to the o Facilitate the spread of infection receptor or cell membrane of the bacteria. DNA-ase, lipase, phospholipase, protease Degrades DNA, lipid, phospholipid The Fc region or fragment crystallizable and protein region is a free are region where the protein A of the bacteria binds. Pathogenesis The reason why the bacteria is easily Adhere to damaged skin engulf is when it undergo opsonization or Liberating toxins opsonization effect however when the protein A binds to the Fc region the Clinical Syndromes opsonization effect disappears and the Cutaneous Infections bacteria exhibit antiphagocytic effect. Folliculitis – inflammation of the fair follicles o A mall red bump or pimple that develops at infection sites of the hair follicle o May extend to the neighboring tissue Sty – is a folliculitis affecting one or more hair follicle on the edge of the upper or lower eyelid Furuncle or boils – deep seated infection, originating from folliculitis o Causes redness, swelling and severe pain o Commonly found on the neck, armpit and groin regions Carbuncle – aggregation of infected Extracellular Toxins furuncles, may form large abscesses Hemolysin, alpha, beta, delta, gamma o Large area of redness, swelling o Dermonecrotic hemolytic and and pain. Punctuated by several leucocidal sites of drainage puss Leucocidin (Panton-Valentine factor) Impetigo – very superficial skin infection o Kills WBC by producing holes in common in children, usually produces their CM blisters or sores on the face, neck, hands, and diaper area Enterotoxin o Acts on the gastrointestinal tract to cause illness, food poisoning K.K. ONG LO | 19  o Characterized by watery bristles, Can cause UTI in elderly hospitalized which become pustules and then men, endocarditis and bacterimia honey colored crust Staphylococcus saprophyticus Deep Infections Normal skin and in the periurethral and Osteomyelitis – inflammation of the bone urethral flora o Bacteria can get to the bone via Common cause of UTI in young bloodstream or following an sexually active women injury o Clinical features: pain, swelling, Specimen Collection deformity, defective healing, in Pyogenic lesion – skin scrapings or swab some case pus flow o Diagnosis: X-ray, MRI, bone Meningitis - CSF aspirates UTI – urine Respiratory infection – sputum Exfoliative Disease Septicemia – blood Exfoliate – scaling off tissues in layers Food poisoning – stool, urine, Suspected carrier - Staphylococcal Skin Scalded Syndrome Previously called dermatitis exfoliative, pemphigus neonatorum, Lyell’s disease Microscopic Examination and Ritter’s disease Epidermal toxin produces by S aureus at skin and is carried by the bloodstream to epidermis, where it causes a split in a cellular level, this toxin separates the outer layer of the epidermis from underlying tissues Toxic Shock Syndrome Cultural Characteristic Caused when TSST liberated by S. aureus Plate reading – describing the petri dish, enters the blood stream to determine if more than 1 organism is Multisystem illness characterized by: present o High fever, headache, vomiting, diarrhea, conjunctival reddening, Nutrient Agar hypotension, skin rashes and kidney failure Staph Food poisoning Caused by enterotoxin A Symptoms: o Nausea, vomiting, severe abdominal cramps, diarrhea, sweating, headache Staphylococcus epidermidis Normal flora of the skin and the mucus Colonies: large, circular, smooth, shiny membrane surface and pigmented. With relatively low virulence Quadrant streaking is done to get a single Caused infection in immunocompromised host growth of organism or isolated colony or Has a distinct predilection for foreign inoculum. bodies (artificial heart valves, indwelling Plate reading is done to determine if there intravascular catheter, CNS shunt, hip is another organism. prosthesis) K.K. ONG LO | 20  Blood Agar Indicator: phenol red and 7 – 10% In blood agar, observe the formation of beta sodium chloride hemolysis. Non-mannitol Staphylococcus Has clear zone fermenters Staphylococcus aureus surrounding the colony. Pink colonies aureus Has beta hemolysis Staphylococcus Mannitol fermenter Staphylococcus No hemolysis epidermidis Yellow colonies epidermidis White creamy colony Staphylococcus No hemolysis saprophyticus White yellow colony Biochemical Test Catalase Test Presence of bubbles ( Staphylococcus aureus positive) Staphylococcus species are catalase positive. o There is a bubble formation because Staphylococcus species is an aerobic bacteria and has catalase enzyme that involves in degradation of hydrogen peroxide into water and oxygen. To do the test, put a hydrogen peroxide on different slide. Then, get a sample from loopful of isolated colony from the plate. Then, emulsify it on hydrogen peroxide. Staphylococcus epidermidis Staphylococcus Positive species Bubble formation Streptococci Negative species No bubble formation Staphylococcus saprophyticus Mannitol Salt Agar For differential media or staining because it contains 2 types of dye. K.K. ONG LO | 21 To Differentiate Staphylococcus Species, Use Novobiocin Test Other Biochemical Test For the differentiation of Staphylococcus Dnase Test epidermidis and Staphylococcus saprophyticus. There is a zone clearing around the streak LONE STREAKING is done and there indicates Dnase activity. should not have space. Uses DNA-Methyl green agar Novobiocin test is applied on plate and POSITIVE: Staphylococcus aureus then incubate. If there is a clearing or zone of inhibition therefore, Novobiocin inhibits the growth of organism because it is sensitive, killed by novobiocin. If there is no clearing or no zone of inhibition therefore, the organism is resistant. Positive, there is a zone of Staphylococcus inhibition or clearing. epidermidis Killed by novobiocin Coagulase Test Negative, there is no zone Staphylococcus Fibrinogen → fibrin (clot formation) when of inhibition or no clearing. saprophyticus the loopful of isolated colony is putted on the Resistant to Novobiocin rabbit plasma. Staphylococcus Positive aureus Staphylococcus Negative epidermidis Staphylococcus Negative saprophyticus Cell Bound Uses slide and observe agglutination in rabbit plasma when the isolated colony emulsifies. Free Coagulase Formation of clot Staphylokinase degrades clot in coagulase test that is why it should be carefully done. K.K. ONG LO | 22  Methicillin resistant Staphylococcus Ensures complete destruction of all aure us microorganisms, including cells, spores S. aureus strain that is resistant to and viruses antibiotics such as methicillin, nafcillin This avoids opportunistic bacteria or any and oxacillin infection in order for an individual not to 3 types get infected. o Hospital acquired (HA) o Ex. equipment in Operating Room, o Community acquired (CA) ER clinic o Health care associated community onset (HACO) Examples of Sterilization Technique Test: Chromogenic test – change of color Dry heat of MERSA colonies within 24-48 hours Autoclaving (100C) using CHROM agar against colorless Gas colonies of non-MRSA Various chemicals Certain types of radiation Disinfection Process that reduces the number of potential disease-causing bacteria and viruses on a material until they no longer present a hazard. Terms decontamination and sanitation means the same thing generally. Disinfectant a chemical used to disinfect inanimate objects. Pasteurization (Example of Disinfection) method of disinfecting liquids (Sterilization & Disinfection) o which should not be exposed for Streaking – by quadrant; to avoid a long time in heat like boiling contamination especially when making wine, Controlling microbial growth is not only beer, milk. Therefore, in the laboratory or hospital but should pasteurization is being also be conducted in our homes. conducted. heat liquids to a certain temperature Death / Killing does not totally destroy pathogens As it relates to microorganism; loss of its ability to multiply under any Decontamination known condition Inactivation or removal of both Ex. UV Radiation of bacteria microbial toxins and the living microbial  pathogens. It will have thymine dimers DNA (base Ex. Attenuated vaccine = inactivating pairing shows thymine to thymine coding) virus to the point where it will not cause  disease but stimulates production of loss of ability of bacteria to replicate antibodies. Sterilization Antiseptic process of removing or killing all Solutions used to disinfect skin and other microorganisms and viruses on or in a living tissues. product K.K. ONG LO | 23  Sanitation Controlling Microbial Growth Reduction of microbial populations to Using Physical Methods levels considered safe by maintaining Methods used to destroy or inhibit public health standards. microbial growth may be physical or chemical. Microbicidal Agents Examples: The suffix -cide or -cidal refers to o Heat “killing”. o Combination of heat and pressure Examples: o Desiccation o Microbicidal agents (microbicides) o Radiation o Bactericidal agents o Sonic disruption o Sporicidal agents o Filtration o Viricidal agents Heat Microbistatic Agents Practical, efficient and inexpensive Drug or chemical that inhibits the method of sterilization. growth and reproduction of 2 Factors that influence effectiveness of microorganisms heat and sterilization: o Temperature o Time Microbistatic Technique Examples: Thermal Death Point (TDP) o Lyophilization (freeze-drying) o Rapid freezing (using liquid nitrogen) lowest temperature that will kill all organisms in a standardized pure culture within a specified period (time). Bacteriostatic Agent One that specifically inhibits the Thermal Death Time (TDT) metabolism and reproduction of bacteria. length of time necessary to sterilize a Some Useful Terms pure culture at a specified temperature. Sepsis Presence of pathogens in blood or tissues Dry Heat Asepsis Absence of pathogens Heating materials in absence of Ex. hand washing; use of moisture. sterile gloves, masks, Simplest method of sterilization gowns; sterilization of Other methods include: surgical equipment; use of o Incineration and flaming disinfectants. surface of metal materials like Antisepsis Prevention of infection forceps and loops. Antiseptic Developed by Joseph Lister (1867) In bacteriology, Alcohol Lamp is being Technique Use of antiseptics used instead of Bunsen burner. Ex. hitting of inoculated loop Moist Heat Boiling or steaming Sterile Technique Reactive water molecules denature Practice of excluding all microorganisms proteins irreversibly by disrupting the from a particular area, so that the area hydrogen bonds. will be sterile. Most pathogens destroyed after 30 minutes of boiling. Boiling is not always effective against some endospores and viruses. Boiling is not an option for thermophiles. K.K. ONG LO | 24 Boiling Structure Of Autoclave: commonly employed but incompletely effective Free Flowing Steam (Arnold Sterilizer) aka Intermittent Sterilization or Tyndallization or Fractionation consist of heating 80 to 100C for 30 mins for 3 consecutive days First Heating vegetative cells and some spores will be killed Subsequent Heating more resistant spores will be killed Pasteurization Process used in food industries for milk Calibration of an Autoclave and other dairy products, fruit juices, wine, beer and other heat sensitive substances. Temperature calibration exposing the product to 62 C for 30 mins Thermocouple is placed into chamber to allow same source location for both Flash pasteurization = 71 c for 15 thermocouples. seconds. Summary: Correlation between the temperature gauge on instrument and autoclave to see Sterilization By Moist Heat if it reaches 100 C Moist Heat Below Pasteurization 100 C Moist Heat At 100 C Boiling Monitoring Autoclave Tyndallization Thermocouple (physical monitoring) Moist Heat Above Autoclave Browne’s tube (chemical) 100 C Autoclave tape (chemical) Spores of Bacillus stearothermophilus (Biological) o Checking if the spores are being Large metal pressure cooker that uses destroyed by the autoclave. steam under pressure to completely destroy all microorganisms. Browne’s Tube Increased pressure raises the temperature is a chemical indicator with a heat of boiling water (i.e above 100 C) sensitive solution within a glass tube, producing steams. which changes from red to green when operated at 121 C; Spores directly the tube is subjected to a high temperature exposed are killed in less than 5 minutes for the required length of time. Normal sterilization time = 10 to 15 mins Autoclave tape to account for variation in the ability of indicate whether a specific temperature steam to penetrate different materials and has been reached. It works by changing to allow a wide margin of safety color after exposure to high temperatures commonly used in sterilization processes, typically 121 C in a steam autoclave. K.K. ONG LO | 25  Spores of Bacillus stearothermophilus change of color indicator Ultraviolet sometimes used to prevent (UV) light or decrease microbial growth. alternative for autoclaving X-rays, gamma may be used to destroy or similar to autoclaving, but using gas for and beta rays prevent microbial growth. some that is sensitive to high temp. Electromagnetic causing DNA to break Ex. Ethylene oxide sterilizer, ETO gas Radiation Cartridge (U.V, X-ray) Ionizing generate electrons, Ethylene oxide Inflammable and potentially Radiation hydroxyl radicals, alters explosive gas DNA, cell death Alkylating Inactivates microorganism agent by replacing labile hydrogen atom on carboxy groups of guanine and adenine in DNA. Often used to sterilize and clean delicate Other alkylating Formaldehyde used without equipment agent pressure consists of tanks filled with liquid solvent Exposure time: 4 to 6 hours and must be (usually water) followed by prolonged period of aeration to Short sound waves are passed through liquid allow gas to diffuse. Soundwaves dislodge organic debris Materials must then be washed and sterilized Ethylene Oxides Sterilizers by another method. resemble autoclaves and expose the loads to 10% ethylene oxide in CO2 at 50 C to 60 C under controlled condition of humidity Filters of various pore size are used to are used to sterilize heat- and moisture- filter or separate larger cells, larger sensitive devices that would be damaged viruses, bacteria and other microbes by pure steam or liquid chemical from gases of liquids in which they are sterilization, including most plastic or suspended. rubber products (Ex. catheters, Paper masks – used to prevent the resuscitation bags, anesthesia, masks, outward and inward movement of most fiberoptic instruments), as well as microbes. non-heat-sensitive devices. Membrane filters – composed of cellulose acetate Gaseous Atmosphere Variables pore size (0.005 – 1 um) Bacteria: pore size (0.2 um) Altering atmosphere of microbes to inhibit growth. not effective for viruses Gas gangrene – caused by Clostridium spp. o Clostridium spp. is anerobic, this is putted to hyperbaric chamber in order to have oxygen and kill anaerobes. metabolic activities slowed most microbes not killed, but growth inhibited Slow freezing – not a good way to preserve bacteriologi