Bacterial Structure & Growth PDF

Enclonar, Kimberly / MLS 3A o Mycobacterium tuberculosis: much Bacterial Structure & Growth...

Enclonar, Kimberly / MLS 3A o Mycobacterium tuberculosis: much Bacterial Structure & Growth granules October 15, 2020 o Corynebacterium diptheriae: BABES ERNST Marx P. Catalan, RMT, MSMT GRANULES o Nocardia spp.: Sulfur Granules Bacteria Unicellular bacteria Endospores Prokaryotic small, dormant, asexual spores that developed Average size: 0.5-2 um in diameter; 2-8 um in length inside bacterial cells Mode of replication: binary fission Produced by: Bacillus & Clostridium o Asexual reproduction Produced in response to harsh environmental o Organism duplicates its genetic materials conditions (DNA) In favorable conditions, the bacteria will be in its o Divides into two parts (cytokinesis) vegetative stage ▪ Each receiving on copy of DNA Mother cell will die and endospore will be released in the environment. They remain dormant. Smallpox virus - smallest virus Thick Protein Coat: makes them highly resistant to: Microscope for viruses - electron microscope o Chemical changes o Temperature changes Bacterial Domains o Starvation and dehydration Archaebacteria o UV and Gamma radiation o Lack peptidoglycan (murein) o Desiccation o Extremophiles Calcium dipicolinate: Found within the core. Makes ▪ Halophiles - loves extreme salt up 10-20% of the spore's dry weight. It maintains concentration the spores: DORMANT STAGE ▪ Thermophiles - loves extreme o Small acid soluble proteins (SASPs): Tightly heat bind and condense the DNA. Responsible ▪ Anaerobic methanogens - loves for resistance to: UV and radiation methane o Can survive for 100 years or more. It is o Not encountered clinical microbiology important in the perpetuation of the Eubacteria species o Includes pathogenic bacteria ▪ Continuity of species o Has tropism to eukaryotic cells o Endospores: Not for reproduction ▪ Tropism - ability of a certain o Fungal spores - used by fungal organisms organism to infect a particular to replicate sexually and asexually type of cell One of the potent virulence factors of organisms. ▪ Proteobacteria o Virulence Factor - components of the Thin gram negative cell organism that contributes to its virulence. wall o Virulence - ability of the organism to cause Includes: Photosynthetic, disease. anaerobic photosynthetic Endospores can germinate in the human body. bacteria and Gram stain: Endospores are colorless (endospore cyanobacteria wall is impermeable to stains and dyes) ▪ Firmicutes Moeller and Schaefer-Fulton stains: Endospores Thick gram positive cell stains wall Used to identify organisms: Includes rods, cocci, o Terminal spores Actinomyctes and ▪ Clostridium tetani Mycoplasma (wall-less o Central spores bacteria) ▪ Bacillus cereus o Subterminal spores Cytoplasmic Structures ▪ Clostridium defficile Do not contain: membrane bound nucleus Starvation initiates sporulation. Genome: single circular chromosome o 0: Decision to use one of two potential Ribosomes: consists of RNA and protein - floating in polar division sites for septum formation the cytoplasm. o 1: Replication and formation of axial o Site of protein synthesis filament o Svedberg unit - sedimentation speed o 2: Septation, 2 unequal compartment Prokaryotic Eukaryotic (forespore and mother cell) o 3: Mother cell membrane engulfs Ribosomes 80S → 60S & 70S → 50S forespore 40S & 30S o 4: Mother cell chromosome destruction. Genome Single linear Cortex is formed. circular o 5: Deposition of coat proteins o 6: Maturation: completing development. Cytoplasmic granules Synthesis of dipicolinic acid. storage deposits and consist of polysaccharides o 7: Mother cell (sporangium) releases spore such as glycogen and lipids such as poly-B- hydroxybutyrate, polyphosphates Cell envelope Structures  Enclonar, Kimberly / MLS 3A Cell (Plasma) membrane o Sieve for water soluble substances to pass Phospholipid bilayer with embedded proteins, does through protein channels called: PORINS not contain: STEROL, except: Mycoplasma spp. ▪ Only allows small molecular o Mycoplasma spp. Does not have cell wall substances to pass through Acts as an osmotic and permeability barrier and ▪ Gram neg are innately resistant to location of the electron transport chain vancomycin Inhibits peptidoglycan Cell Wall Structures layer Humans do not have cell walls Vancomycin cannot pass Rigid structure, provides shape and rigidity through porins and Two major types: cannot reach the thin o Gram positive cell wall peptidoglycan layer in o Gram negative cell wall periplasmic space Prevents bursting of cells from the high osmotic o Attachment sites to host cells pressure inside Periplasmic space: contains the thin peptidoglycan o Has a gel-like matrix containing nutrient Gram Positive cell Wall binding proteins and degradative and Thick peptidoglycan (murine) layer. detoxifying enzymes Does not have inner and outer membrane o Contains amino acids, sugars, vitamins, Each layer consists of 2 alternating polysaccharides: enzymes, irons, and essential nutrients o N-acetyl d-muramic acid (NAM) o N-acetyl d-glucosamine (NAG) Acid fast Cell Wall Each layer is cross-linked (peptide bridge) with short Acid fast organisms are generally: gram positive cell amino acids peptides consisting of 4 amino acid wall residues. Contain a waxy layer of glycolipids and fatty acids o L-alanine known as: MYCOLIC ACID, bound to the exterior of o D-alanine the cell wall o D-glutamate o 60% of the cell wall is lipid o Meso-diaminopimelate o Strong hydrophobic molecule, making the Teichoic acid: anchored to peptidoglycan organism difficult to gram stain (horizontal) ▪ Gram ghost Lipoteichoic acid (LTA): anchored to cell membrane o Resistant to: acids thus the name acid fast Present in acid fast bacteria such as: Gram Negative cell wall o Mycobacteria spp. & Nocardia spp. Two layers: Inner and outer membrane Outer membrane contains: proteins, phospholipids Cell Wall Deficient bacteria and lipopolysaccharide (LPS) Mycoplasma spp. o LPS: main component of gram neg cell wall Ureaplasma spp. o LPS has 3 regions: o Contain sterol in their cell membrane ▪ Antigen O polysaccharide o Pleomorphic It can bind to antibodies ▪ Core polysaccharide Surface Polymers ▪ Inner lipid A known as the: Glycocalyx ENDOTOXIN Network of polysaccharide that project from the Responsible for: fever cellular surfaces of bacteria and shock conditions Found external to the cell wall of the organism For it to function: cell Not common to all should be lysed, and once Capsule it is liberated, it o Made of polysaccharide polymers or stimulates the immune polypeptides response of the host o Compact & tightly associated in the cell Causes uncontrolled o Role in virulence - evasion of phagocytosis inflammation o Can be removed by boiling bacterial There could be suspension hemorrhage o Hydrophilic Reduced RBCs circulating o Gram stain - appears as clear halo to organs → reduced o Capsular staining - Hiss & Anthony's stain oxygen → shock Slime layer Even if the organism is o Similar to capsules but are more diffuse dead, endotoxin still layers surrounding the cells functions o Made up of polysaccharides o Dosage of antibiotic is important to o Role in virulence - evasion of phagocytosis prevent endotoxemia o Used by organisms to attach with each o Test to detect endotoxins in medical other or to attach to host tissues equipment: limulus amebocyte lysate Mechanisms of Phagocyte Evasion (LAL) test o Masks attachment sites of phagocytes and ▪ Horseshoe crab, Limulus other components of the immune polyphemus response o Barrier for hydrophobic compound  Enclonar, Kimberly / MLS 3A ▪ Pathogen-associated molecular ▪ Flagella surrounding the entire cell patterns (PAMPs) - components in o Amphitrichous the cell wall of the organism that ▪ Taft, or bundles of flagella at both can be recognized by the poles phagocyte o Atrichous Unique to bacterial cells ▪ Absence of flagella Danger signals ▪ Endocytic pattern-recognition receptors (PRR) - attachment sites of PAMPs ▪ Direct interaction - direct interaction between PAMPs and PRR ▪ Indirect interaction - PAMPs and antibody + PRR ▪ Capsules are weak PAMP o Molecular mimicry. ▪ Components of capsule that are Flagellar stains similar to host's "self" o Fisher & conn components o Leifson & Gray ▪ S. pyogenes has a hyaluronic acid capsule similar to human Pilus/Pili connective tissue Non-motile, long, hollow protein tubes that o Difference in the nature of the phagocyte's connects two bacterial cells membrane and the bacterial capsules. Also known as: conjugation pili Bacteria must be more hydrophobic than Role on virulence: mediate DNA exchange (i.e, DNA the phagocyte. containing antimicrobial resistant genes) Some Killers Have Pretty Nice Capsule o Streptococcus pneumoniae (and group B Fimbria/Fimbriae Strep) Non-flagellar, sticky, proteinaceous appendages o Klebsiella pneumoniae Use by organisms to adhere to one another and to o Haemophilus influenzae the environment o Pseudomonas aeruginosa Role in virulence: Adherence to host tissues o Neisseria meningitidis o Crytococcus neoformans - not a bacterium, Fundamental shapes it is a fungus Bacilli (bacillus) - rod shaped organisms Mucoid colonies on culture media indicate presence Cocci (coccus) - spherical or round organisms of encapsulated bacteria Spirilla (spirillum) - spiraled or comma shaped Bacterial Arrangements Two factors that affect bacterial arrangements: o Plane of division o Position taken after cell division ▪ Depends on plane of division Sagittal plane o Diplococci or cocci impair o Streptococci Sagittal and frontal o Tetrad Flagellum/Flagella Sagittal, frontal, and transverse Organ of locomotion o Sarcinae Made up of flagellin proteins Variation in the possession of flagella Chemotactic agents - promotes chemotaxis Responds to chemotaxis o Positive chemotaxis ▪ Movement of organisms towards the source of the signals ▪ Favorable conditions o Negative chemotaxis ▪ Movement of organisms away from the source of signals ▪ Harmful conditions Location of flagella o Monotrichous ▪ 1 flagellum at 1 pole o Lophotrichous ▪ Bundle of flagella at 1 pole o Peritrichous  Enclonar, Kimberly / MLS 3A Pairs o Nitrite o Diplococci - N. Gonorrheae o Ammonium (NH4+) o Diplobacilli - MTB Most microorganisms use NH4+ as a sole nitrogen Chains source o Streptococci Many organisms possess the ability to produce it o Streptobacilli from amines Grapelike clusters o Staphylococci Growth Factors Groups of 4 - tetrads Organic compounds needed by bacteria in order to o Peptococcus grow. In bacterial culture, these substances are Packets of 8 - cuboidal usually provided in the culture medium. o Sarcinae o Prototrophic - do not require an exogenous Palisades source of growth factor since they o Organisms tend to place themselves side synthesize their own by side (Corynebacterium) o Auxotrophics - require the addition of growth factor to culture media for growth Growth to occur. Orderly increase of all chemical constituents of the Fastidious organisms - requires additional nutrients cell Examples: A process which entails the replication of all cellular o B-complex vitamins structures, organelles and protoplasmic o Amino acids components from the nutrients present in the o Purines surrounding environment o Pyrimidines Under favorable conditions, almost all bacteria are o Pentoses able to produce very rapidly o Fatty acids Generation time/doubling time o The average time required for an organism Inorganic salts to double its number Salt in small amount stimulates the growth of some ▪ E coli - 20 mins organisms Organisms requiring concentration are called 3 Major Nutritional Needs halophilic Source of Carbon 2-30% NaCl o For making cellular constituents o Carbon represents 50% of the dry weight Oxygen Requirement of a bacterium Aerobes - grow in the presence of atmospheric A source of nitrogen (free) oxygen o For making proteins o Obligate aerobes: grow only in the o Nitrogen makes up the 14% of the dry presence of oxygen (15-21% O2, 1% CO2) weight ▪ Makes ATP in the presence of Source of energy (ATP) Oxygen o For carrying out cellular functions o Facultative anaerobes: fundamentally aerobes, but can grow in the absence of Carbon Requirement oxygen Classification of organisms ▪ Makes ATP aerobically with o Autotrophs (lithotrophs) - use CO2 as Oxygen, but can switch to carbon source, with only water and fermentation if oxygen is absent inorganic salts as substrates o Microaerophiles: grow best at low or ▪ Phototrophs - obtain energy reduced oxygen tensions (2-10% O2) photosynthetically Anaerobes - grow in the absence of atmospheric ▪ Chemilithotrophs - oxidation of oxygen inorganic compounds o Obligate anaerobes: grow only in the o Heterotrophs (organotrophs) - require an absence of O2. organic source of carbon (ex. Glucose) and o Aerotolerant anaerobes: do not grow well obtain energy by either oxidation or but do survive in the presence of O2 fermentation however, they cannot perform metabolic ▪ All bacteria that inhabit the processes unless they are place in an human body belong to this oxygen-free environment category Why are some organisms anaerobic? o These are toxic end-products Nitrogen Requirement Major component of proteins and nucleic acids of a typical bacterial cell Nitrogen fixing bacteria - organisms that can directly use atmospheric Nitrogen gas (N2) Nitrogen assimilation - formation organic nitrogen compounds like AA from inorganic nitrogen However, aerobic organisms have the enzyme compounds in the environment superoxide dismutase and catalase o N2 o Nitrate  Enclonar, Kimberly / MLS 3A CO2 Some organisms require a higher concentration (5- 10%) of CO2 for growth Capnophiles Moisture This is indispensable for bacterial growth. It serves as a solvent for food and forms the major portion of the protoplasm. Organisms requiring increased moisture content are termed humidophiles. Temperature Determination of Cell Numbers Every bacterium has an optimal temperature: the Direct Counting Using the Microscope temperature at which the organism grows best Used to estimate the number of bacteria in a Psychrophilic (cold loving) - grows at 1C-20C suspension Mesophilic - grows at 20-40C (most pathogens grow Cannot distinguish live and dead cells at 37C) Uses: Petroff-Hausser Counting Chambers Thermophilic (heat loving) - grows at 50-60% pH Most pathogenic bacteria have an optimal pH of 7.2-7.6 Acidophiles - grows at pH 6.5-7.0 Neutrophiles - grows at pH 7.5-8.0 Alkalophiles - grows at pH 8.4-9.0 Osmotic pressure Organism requiring high osmotic pressures are called osmophiles. Bacterial Growth curve Refers to the increase in number of organisms Is obtained by plotting the logarithm of the number of cells against the time of growth Direct Plate Count/Serial Dilution Lag phase/period of rejuvention/Physiologic youth Growing dilution of bacteria on agar plate to o The period of adaptation of the organisms determine the colony forming units per ml (cfu/mL) to their new environment characterized by Provides a count of viable organisms only little or no multiplication o Best observed: after 6 hours of incubation ▪ Required incubation period: 18-24 hours o The cell sin this phase are very active metabolically. Active synthesis of enzymes and other essential constituents occurs. Exponential/Logarithmic Phase o 18-24 hours after incubation o The period at which the cells are in a state of balanced growth characterized by maximal rates of cell division and mass increase. o It is during this phase that the generation Density Measurement time is constant. Testing for turbidity for bacterial suspension Stationary/Plateau/Equilibrium phase McFarlan Standard o 48hrs Measured using Optical Density (OD) o The period at which the rate of cell o OD at 600nm or short OD600 production equals the rate of cell death. o Growth ceases due to: Bacterial Cultivation ▪ Accumulation of waste products The process of growing microorganisms in culture ▪ Exhaustion of nutrients by taking bacteria from the infection site (in vivo ▪ Change in pH and other factors environment) Death/Decline Phase Growing them in an artificial environment in the o 72hrs or more laboratory (in vitro) o The period at which complete cessation of multiplication occurs such that the death Agar rate in the medium increase rapidly. Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae: Gelidium amansii Makes culture medium solid  Enclonar, Kimberly / MLS 3A Microbial growth does not destroy the gel structure ▪ Alkaline Peptone water (APW) because most microorganisms are unable to digest ▪ Selenite F broth agar ▪ Tetrathionate broth o Stool samples Basic Ingredients of Culture Media Enriched Media Beef Extract o Addition of extra nutrients such as blood, o Beef is boiled and the resulting broth is serum, egg yolk, vitamins and growth concentrated into a paste or dried to a factors to basal medium powder. It contains amino acids, vitamins o To promote the growth of fastidious and minerals. bacteria Peptone ▪ Blood agar o Protein is converted into proteoses, ▪ Chocolate agar peptides and amino acids by means of ▪ Modified Thayer Martin Agar enzymatic hydrolysis. o How many percent of blood agar or o Source of energy for those that cannot chocolate agar are usually prepared in the utilize CHO laboratory? 5-10% blood Yeast Extract o What are the sources of blood used in the o This is the water soluble portion of preparation of BA and CA? Most common autolyzed yeast and contains amino acids, is sheep's blood. Can also use rabbit, nucleic acids, vitamins and other growth horse or human blood, or reagents such as factors. 2% Hgb. o How to prepare? CHOC is heated to lyse Culture Media: Classification RBCs. Therefore, hemolytic patterns According to Consistency or Physical State cannot be observed, Liquid/Broth Media Selective Media o No agar/solidifying substances o Inhibits commensal and contaminants to ▪ Tryptone Soy Broth (TSB) promote growth of a particular organism ▪ Thioglycolate Broth o Contains inhibitors ▪ Nutrient Broth o What is swarming of proteus? Cocentric o Indication of growth: turbidity rings of growth that are formed as cyclic Solid Media events of swarmer cells. o Contains 2-3% agar to solidify ▪ Plate Media BA, CA, MAC ▪ Tubed media TSI, LIA, citrate tube Semi-Solid Media o Contains 0.2-0.5% agar ▪ SIM (sulfide, indole, and motility medium) Biphasic Media o Comprise both solid and liquid medium in the same bottle ▪ Castaneda biphasic system - Brucella According to Composition Synthetic Media o Chemically defined Non-synthetic media o Not chemically defined o Example: BA Tissue Culture Media o With living cells o For viruses o Intracellular bacteria According to Use Selective & Differential Media Basal/Simple Media o Selective media & differential media - o Allows most non-fastidious organism to grows particular organisms and grow differentiates its biochemical ▪ Nutrient Agar characteristics ▪ Nutrient Broth o Contains: Enrichment Medium ▪ Inhibitors o Liquid media that inhibit commensals and ▪ Carbohydrates promote the growth of a particular ▪ pH indicators organism o Blood Agar for Hemolytic Patterns o Used to recover pathogens in an anatomical site rich with flora  Enclonar, Kimberly / MLS 3A ▪ Beta hemolysis: Complete Distribution hemolysis characterized by a clear o Distribute in test tubes zone around the colonies Sterilization Although generally, non- o Autoclave/Oven selective but can be Note: Plated medium - sterilized first before distribution enriched with Colistin- Nalidixic acid for Inoculation of Culture Media inhibition of gran Liquid Media negative bacteria o With the use of a sterile Pasteur pipette ▪ Alpha hemolysis: incomplete o Inoculate by shaking a previously heated hemolysis characterized by wire loop or needle. greenish pigmentation of the Slant Media colonies o Zig-sagging manner across the entire ▪ Gamma hemolysis: no hemolysis surface toward the mouth of the tube o Basic Principle: CHO utilization media as Slant & Butt Media example o Inoculate the butt first by stabbing the ▪ Inhibitor needle to the bottom of the medium and then streak the surface in a zigzagging manner toward the mouth of the tube. Butt Media o Stab the medium with an inoculating needle Plated Media General purpose Isolation Streak 1. To yield a semi-quantitative grow 2. Specimen is applied by rolling a swab or placing a drop of liquid on a small area at the edge of the plate 3. Inoculating loop is sterilized and allowed to cool thoroughly before streaking 4. Cooled loop is passed back and forth through the inoculum in the first quadrant several times 5. First quadrant should be at least a quarter of the plate, and the streak line should be closed together 6. The plate is turned, and quadrant 2 is streaked by passing the loop through the edge of the first quadrant a few times and then streaking the whole 2nd quadrant 7. Repeat step 2 but now in the 3rd down to the 4th quadrant Blood agar - non-selective. But can add nutrients to become selective. Special Culture Media o Specially prepared to support the growth Quantitative Isolation Technique of specific microorganisms To determine the number of bacteria present and decide if the isolate is a true infection or a contaminant Used for urine specimens 1. Urine is mixed well, and a calibrated loop (0.1 or 0.001 mL) inserted in the urine and transferred to the culture medium by making a single streak down of the center of the plate 2. Without flaming, the loop is streaked back and General Steps in the Preparation of Culture Media forth into the original inoculum Weighing Formula o Weigh the different ingredients and then Colony count = # colonies x factor place in a clean, dry containers. o 1ul (0.001ml); factor 1000 Dissolving o 10ul (0.01ml); factor 100 o Add the exact amount of solvent to the If a 1ul is calibrated loop yields 145 colonies, the ingredients and then dissolve by heating colony count is 145,000 colonies/ml urine Titration >100,000 = infections o Adjustment to the right pH (pH 7.2) 1000-100,000 = contaminants  Enclonar, Kimberly / MLS 3A Semi-quantitative Maki Roll Plate 1. Used to culture catheter tip specimens to diagnose catheter-related blood stream infection 2. Catheter is rolled back and forth onto a blood agar plate 3. After incubation, count the colonies o >15 CFU on the plate: TRUE INFECTION o