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UC / Biology / BIOL 4011 / What is the meaning of colony formation?

What is the meaning of colony formation?

What is the meaning of colony formation?

Description

School: University of Cincinnati
Department: Biology
Course: Microbiology
Professor: Dennis grogan
Term: Fall 2019
Tags: Biology
Cost: 50
Name: Exam 4 Study Guides
Description: These are the filled out outlines for Exam 4!
Uploaded: 11/29/2019
10 Pages 49 Views 9 Unlocks
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Outline 19 concepts: natural microbial communities, complexity-- isolation of  individuals; microscopic & molecular analyses -------------  


What is the meaning of colony formation?



 in nature, micro-organisms occur in communities > very large, complex,  dynamic  

 challenging to identify  

o i) the members of these communities

o ii) what each contributes  

 historical emphasis on isolation in pure culture 

o 1. choose environment (inoculum)  

o 2. inoculate appropriate media under appropriate conditions  (enrichment)  

o 3. streak on corresponding solid medium to get colonies (isolation)   the environment (community) may include thousands of species;  conventional cultivation yields only those that  

o i) reproduce under the chosen growth conditions

o ii) reproduce faster than all the others  

 The “enrichment” conditions used have a critical impact on which  community members are cultured


What is the meaning of fluorescent staining?



o classic example = Azotobacter  

 proved that it is possible to fix N2 aerobically  

o micro-cosms can also enrich for certain bacteria; example of  Winogradsky column  

 see characteristic structuring of communities along vertical  gradients, reflecting complementary metabolism  

 colony formation is most common route to pure culture

o streak plate, dilution, pour plate, agar deeps  

o more difficult alternative: dilution to extinction in liquid  

 newer methods involve physical separation of individual cells (optical  tweezers, FACS)

 Alternatively, can use high-throughput approaches to create many  enrichment conditions in parallel  

 Non-culture techniques  


What is the meaning of fluorescent in-situ hybridization (fish)?



o fluorescent staining: need a signal that is specific to cells  (overcomes background of matrix)  If you want to learn more check out How to develop a marketing strategy?

 DNA stains (DAPI, Acridine Orange, SYBR Green)

 permeability stains (live/dead)

o higher specificity provided by fluorescently tagged nucleic acid:  Fluorescent In-situ Hybridization (FISH)

 procedure: fixing, permeabilization, hybridization to rRNA,  

washing  

 result: staining that reveals morphology, phylogenetic  

groupings, physical associations  

 PCR Methods  

o PCR can amplify a short sequence from many members of complex  communities

o Detection can be very sensitive and informative; most common target  = SSU rRNA genes

 rRNA sequence serves as a ‘tag’ or provisional ID for uncultured organisms  (“OTU” or “phylotype”)  

 Microarrays test for the abundance of particular rRNA (or other gene)  sequences  

o whole-community DNA is amplified, labeled (fluorescent), and  hybridized to thousands of probes  

o Increasingly common for rRNA amplicons to be identified directly by  DNA sequencing

 Metagenomic methods are increasingly feasible (brute-force sequencing of environm. DNA) If you want to learn more check out What is the meaning of marasmus?

o often possible to assemble reads into nearly complete genomes   Comparing the environmental sequences with those from known species  provides information about the metabolic capabilities of species within the  microbial community

Outline 20 concepts: ecology of micro-organisms trophic interactions, spatial scales  communities that form on surfaces -------------  

 micro-organisms in nature illustrate fundamental principles and phenomena  of ecology, but with some important differences  

 micro-habitats, micro-environments  

o habitat may be structured on very small spatial scales  We also discuss several other topics like What is the meaning of secondary effects in economics?

o cells can be densely packed, influence local environment  

 mass vs. energy  

o ecosystems require net inputs of energy, but recycle much of the  nutrients within them  

 microbial ecosystems may be small (Winogradsky column)  

 metabolic complementarity  

o trophic structure often based on consumption of metabolic products,  not of organisms  

o “waste chain” instead of food chain  

 resilience  

o microbial populations are huge (may have trillions of individuals)  o natural environments yield “feast and famine”; communities rebound  quickly  

o stress changes the abundance of species, but not species richness  o How do u prove that microorganisms are extinct We also discuss several other topics like Who made the holy trinity with virgin st. john and donors?

 Huge population  

 Survival forms (ex. Endospores)

 Adapted to “feast and famine” existence  

 MICROBIAL COMMUNITIES in the environment  

o Biofilms  

 mixtures of bacterial cells, embedded in gelatinous matrix  

(polysaccharide) on a solid surface  

 Not mono cultures, not random either

 result of a colonization process with several steps  Don't forget about the age old question of What is important when establishing an overall strategy or objectives?

 potential advantages for micro-organism:  If you want to learn more check out What are the common types of solutions?

 nutrition (filter-feeding), protection, beneficial interactions

 Steps

 Cell attachment

 Altered metabolism  

o Secreting polysaccharides- capsule

 Colonization

 Enlargement: more complex w bubbles and channels  

 Benefits

 Diff species can live together if they can help each other

 Protection in large numbers/upper layers protect lower  

layers  

o Bacteria in biofilm harder to kill than in liquid

 Allows microorganisms to be a filter feeder

 Cons/problems

 Surgical/medical implants (form biofilms)

 Cystic fibrosis

 Water systems, bigger problems in hospitals  

 Symbioses not always beneficial to both species  

o Microbial mats  

 visible strata representing different species and guilds  

 illustrate function of a microbial ecosystem: net input of energy,  but recycling of nutrients within mat  

 Usually supported by autotroph

 Thicker than biofilms (niches)

 Vertical diffusion

 Trophic interactions  

 [Discussion question: properties of microbial communities related to  symbioses with plants or animals]

Outline 23: mutualisms involving micro-organisms complex interactions, microbial  contributions to host nutrition  

 Mutualism with plants  

o legumes grow well without N fertilizer, due to symbiosis with N2  -fixing bacteria  

o the bacteria are broadly termed ‘rhizobia’(span many divergent  bacterial groups)  

 Most nitrogen-fixing bacterial symbionts of plants

o host-bacterium specificity results in “cross-inoculation groups”  Group consists of all legumes that can be infected by particular  rhizobia  

o the bacterium infects the plant to form a root nodule  

 represents major steps  

 1. recognition between root & bacterium, attachment  

 2. bacterium secretes Nod factors  

o Nod factors: induce root hair curling and trigger  

cell division

 3. bacterium enters root hair  

 4. infection thread helps bacterium penetrate root  

 5. bacterial multiply, convert to bacteroids  

o Bacteroids: plant + bacteria transformed together

 6. root encases bacteriods in nodule, they fix N2  

o early steps involve various signals and responses of both bacterium  and plant root  

 Plant root: adhesion molecules (lectins), flavonoids that  

induce bacterial Nod genes  

 Bacteria: ‘rhicadhesin’, nodulation factors

o In the nodule, the plant provides: carbon compounds (C4 organic  acids), O2 buffering (leghemoglobin)  

 bacterium provides: NH3, glutamate, glutamine, etc.  

 Mutualism with animals  

o alimentary canal is a logical habitat for micro-organisms  

 omnivores, carnivores are monogastric (acidic stomach followed  by intestine)  

o herbivores have additional digestive organs  

 ‘foregut fermenters’ have rumen (before the stomach)

 hindgut fermenters have caecum after the stomach  

o Ruminant nutrition features microbial degradation of plant material in  the rumen

 reticulum passes fine material to omasum, then abomasum  for chemical digestion  

 digest moves to small and large intestine for absorption  

o in the rumen, cellulolytic bacteria (Fibrobacter succinogenes,  Ruminococcus alba) produce cellulases  

 the cellulose is hydrolyzed to glucose, and the glucose is  

fermented to succinate and lactate; these are fermented to  

short-chain FA

 CO2 and H2 are also formed, but are converted by methanogens to CH4  

o End result: animal absorbs VFAs (acetic, propionic, butyric), belches CO2 , CH4

 the microbial biomass is digested by the omasum and  abomasum  

o the rumen community is complex and its exact composition depends  on diet  

 abrupt changes can be fatal (example: rapid shift to high-starch  diet - excess lactic acid)

Chapter 24 : normal human microbiota, phylogenetic composition, variation among  body sites ----------

 human body provides habitat for many bacteria (outnumber human cells)  o normal microbiota: the microbial species normally associated with  healthy people  

 the communities are complex, mostly bacteria; vary among individuals, body  sites, over time

o normally do not cause disease; usually exclude pathogens, influence  host physiology  

 DNA analyses reveals complex community of phylotypes -  

“microbiome”  

 GI tract  

o humans (omnivores & carnivores) have simple system: monogastric  (one stomach)  

o bacterial density and community complexity increase along tract;  nutrient and oxygen concentrations decrease 

o stomach secretions are strongly acidic (= antimicrobial), but mucosa  provides some protection for bacteria  

 gastric microbiome has few species; many people (~half) are  colonized by H. pylori  

o considered opportunistic pathogen; promotes chronic inflammation,  can lead to ulcers or cancer  

o small intestine  

 duodenum still acidic  

 jejunum transition to neutrality  

 ileum transition to anoxia  

o large intestine  

 cecum transition  

 colon anoxic, high bacterial content  

 lumen is mostly bacteroidetes & Gram-positives (>1010/g);  have digestive functions

 Oral cavity 

o saliva continually washes surfaces, contains lysozyme &  

lactoperoxidase

o infant gums are first colonized with aerotolerant Gram (+) bacteria  (streptococci, lactobacilli)  

o eruption of teeth leads to appearance of anaerobes (tooth surfaces and gingival crevice) Veillonella, Fusobacterium 

 Respiratory tract 

o moist surfaces with antimicrobial defenses (mucous flow, secreted Ig)  o upper  

 (nostrils, nasopharynx, pharynx)  

 colonized by staphylococci, streptococci, diphtheroids and G(-)  cocci  

o lower  

 (lungs, bronchi, trachea)  

 nearly sterile due to action of ciliary escalator

 Urogenital tract 

o urine is initially sterile; bladder and proximal urethra also normally  sterile, while distal urethra is colonized by facultative aerobes (shorter  in women, more prone to cystitis)  

 Proteus  

o vagina normally colonized by complex communities that respond to  female physiology  

 pre-pubescent/post-menopausal: (neutral pH) staphylococci,  streptococci, diphtheroids, E. coli  

 reproductive: glycogen produced, fermented by Lactobacillus  -> lactic acid ->low pH  

 16S data: 5 types of vaginal communities common among adults (defined by dominant L. species)  

 Skin  

o functions as antimicrobial barrier, but properties vary among body  sites (moist vs. dry vs. oily)  

 moist: corynebacteria, staphylococci  

 dry: beta-proteobacteria, flavobact.  

 oily: propionibacteria

o bacterial colonization discouraged by low moisture content, salt,  sebum  

 Salt excretion from sweat

 Sebum: oil secretion; bacteria can produce fatty acids to keep  other bacteria away

o sebum degrades to produce fatty acids - toxic to many bacteria   disorders involving the human microbiota 

o IBD: inflammation due to immune system reacting to some species of  the gut microbiota

 (the exact etiology remains unclear, and several factors  

contribute (diet, environment))  

o obesity: germ-free vs. normal vs.genetically obese mice implicate  microbiota (esp. methanogens)  

 40% leaner (underweight) than normal mice

 Not getting enough nutrition from their food

 Normal mice get 10% caloric intake from volatile fatty acids  (VFA)

 Similar to rumen having animals  

 Genetically obese mice have more methanogens in colon than  normal mice do

Outline 25 concepts: requirements and stages of pathogenesis, virulence factors,  diverse roles of toxins in disease ----------------------  

 Pathogenesis  

o Only a small minority of bacteria cause disease.  

o Central question: what makes the pathogen special?  

o important terms  

 infection: a microbial population outside the normal microbial  sites (often with inflammation)  

 virulence: the relative effectiveness of a pathogen (often  

quantified by LD50) (contrast with pathogenicity)  

 opportunistic pathogen: causes disease only in compromised  individuals  

 tropism: certain pathogens focus in particular tissues or organs   Typical infection process

o Adherence

 non-specific (example: S. mutans in dental plaque; adherence  mediated by bacterial dextran)  

 specific (example: N. gonorrhoeae known to colonizes urethra,  specific binding between bacterial surface protein “Opa” and a  

host-cell surface protein CD66)  

o after the pathogen has successfully adhered to a site, it must colonize  it, followed by spreading  

 through bloodstream: bacteremia vs. septicemia  

 through tissue: invasion

o invasion is often aided by diverse virulence factors  

 (example: capsule (discourages phagocytosis))  

o other virulence factors function as digestive enzymes

 (hyaluronidase, streptokinase, coagulase)  

o other soluble proteins kill or impair host cells: “exotoxins”  

 three broad classes (cytolytic, AB, superantigen); may or may  not be part of the invasion process  

 cytolytic exotoxins include hemolysins (blood agar plate,  

blood as indicator)

 AB-type exotoxins have two subunits or fragments  

o diphtheria toxin: B portion binds a cell receptor, A is

cleaved off, enters host cell cytoplasm, ADP

ribosylates EF-2  

 tetanus and botulism toxins are also AB toxins; act by  

blocking neurotransmitter release  

o Tetanus

 Acts on modulatory neurons  

 Glycine released as neurotransmitter  

 Cl. Tetani- obligate anaerobe

 Leads to complete uncontrolled contraction  

of muscle

o Botulism

 Cl. Botulinum

 Blocks release of acetylcholine

 Lose ability to contract muscle  

o Gram(-) bacteria also have “endotoxin” - the LPS that forms the outer  layer of the OM  

 endotoxin normally released only from cells that die & lyse   if LPS enters bloodstream, it triggers cytokine release, causing  fever, inflammation  

o >any fluid added IV must be screened for LPS; standard assay (LAL)  uses blood from horseshoe crab

 virulence genes  

o early genetic studies of pathogens focused on finding the differences  that made certain strains virulent  

o typically find genetic identity to non-pathogens punctuated by dense  clusters of virulence genes  

 “pathogenicity islands”  

o these modules can be transmitted between different species; thus  observe similar pathogenicity in different bacteria that are not closely  related

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