EXAM: general part Flashcards
- Position and significance of bacteria in the biosphere.
Pathological importance:
- Saprophytes:
*Some very useful such as in intestine and in rumen. Their metabolic activity helps us to utilize nutrition, digest and produce vitamins.
*Also in monogastric animals eg. Eq, Su, rabbit which have large caecum where the bacteria break down and utilize cellulose.
- Pathogens: only a small part of all bacteria. Affect human, plants and animals.
*Difficulties for a pathogen when colonizing a host: limited oxygen, limited Fe, toxic radicals produced by infected cells, immune system and body temperature.
Habitat, importance:
- Large part in the balance of the ecosystem, by having a key role in break down and recycle of organic matter.
- Wide spread in the environment.
16rRNS
- Archeobacteria, most ancient forms, all are saprophytes.
- Eubacteria, most are saprophytes but some are pathogenic.
- Eucaryotes (Plants, Algae, Fungi, Protozoa, Animals)
- Eukaryotes and prokaryotes.
Eukaryotes and prokaryotes:
- Prokaryotes: most simple organisms and consist of a single cell.
- Own metabolism, can produce energy.
- Main characteristic: no nuclear membrane. Nucleus is free in the cytoplasm.
- No cell organelles except ribosome which is smaller in size. Single chromosome and a ring-like plasmid.
- Not as structured as eukaryotes.
- Muramic acid in cell wall. - Characteristic - look at the table
- The size and shape of bacteria
Morphology:
- Shape:
- Rod (bacillus) (eg. Bacillus, Actinomyces, Clostridium, Corynebacterium, Listeria)
- Cocci (spherical) (eg. Streptococcus, Staphylococcus)
- Helical (spiral) (eg. Brucella, Campylobacter, Helicobacter) - Size: 0,2-100 (500) μm
- Rod 0.5-10 μm
- Cocci ~ 1 μm
- Helical 0,2-100 μm - Arrangement:
- Single
- Chain (eg. Streptococcus, Bacillus)
- Cluster (eg. Staphylococcus)
- Palisade
- Their examination by light, dark-field, phase contrast, fluorescence and electron microscope.
LIGHT Microscopy
- Visible light 400-700nm
- Resolving power (0.2 mm)
- Immersion objective (oil)
- Magnification (1000-1500x)
DARK-FIELD microscope
-Special condenser
-Illuminate with oblique ray
-Light, dark background
-Corpuscular elements (bacteria) are glittering
-Examination of bacterial motility
PHASE-CONTRAST microscope
-Optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image.
-Phase shifts become visible when shown as brightness variations.
FLUEROSCENCE microscopy
-Optical microscope that uses fluorescence and phosphorescence instead of/in addition to, scattering, reflection, and attenuation or absorption
-Study properties of organic or inorganic substances.
ELECTRON MICROSCOPE
-(transmission, scanning)
-Uses a beam of accelerated electrons as a source of illumination.
-Higher resolving power than light microscopes and can reveal the structure of smaller objects.
- The structure of bacterial cell, cell wall
Essential cell components: - Cell wall - Cytoplasmic membrane - Cytoplasm - Nuclear material Non essential cell components: - Capsule - Flagella - Fimbria - Spore Cell wall: - Except Mycoplasma - Protection: mechanical effect, osmosis - Transport: non selective permeability - Peptidoglycan - N-acetylglucosamine (G), N-acetylmuramic acid (M) - Peptide units, peptide bridges: how the layers of G and M are vertically connected. - Lyzozyme: cleaves bw. G and M - Gram positive: teichoic acid, carbohydrates, proteins, lipoids, vaxes. - Gram negative: lipopolysaccharide complex (LPS), porins.
- The structure of bacterial cell, cell wall –> (talk about gram positive and negative)
Gram +:
- All “M”- units are in close connection with peptide bridges, 3D cross-connection.
- Cell wall consists of 10- 12 layers, with “ empty” spaces (teichoic acid, carbs, proteins, lipoids, vaxes) which fills up with dye blue color.
- Techoic acid and polysacharaides are the most important antigens of Gram + bacteria.
Gram -:
- Basic structure is the same, but there is a few differences from the G+ bacteria:
*Only 1/3 of “M-M”- units are in close connection less or no peptide bridges.
*There are only two layers.
*The outer membrane is a so called LPS. With a uniform layer of lipids, wich prevent penetration and excretion of materials both in and out of cell. There are pores in the membrane, porines. Therefor the Gram– are harder to penetrate and for AB to work on.
- Between the sidechains there is a polysaccharide core.
The “O”-specific side chain is responsible for specificity of bacteria endotoxins.
- The core is hydrophobic.
- Cytoplasm, cytoplasmic membrane, nuclear material.
Cytoplasm:
- Ribosomes 70S (30S, 50S), 25nm. Smaller than ribosomes of hostcells, important in treatment w. AB, which only attacks the smaller ribosomes of bacteria.
- Inclusions (starch, glycogen, polyphosphate).
- Lipid granules.
- Protein synthesis.
Cytoplasmic membrane:
- Barrier, transport (nutrients, waste material), 5-10 nm - 2/3 protein, 1/3 lipoid, phospholipids, no sterols
- Enzymes
- Mesosoma
- Protoplast – spheroplast, if cell wall disappears. Gram+:
protoplast. Gram-: spheroplast.
- It separates cytoplasma from environment and are important in transportation.
- Energy production in membrane vital.
- Important in the metabolism of the cell.
- To increase the surface of the membrane there can be invaginations, due to the E-prod, the surface does not always cover need of bacteria -> invagination -> incr. surface -> incr. E prod.
Nuclear material:
- Nuclear membrane is missing.
- Nuclear material in the cytoplasm.
- Single chromosome, haploid, ring form, superhelix. The size of the chromosome is large compared to bacteria cell. The length is ≈1-2 mm, compared to cell which is only a few μm. The enzyme which produce the superhelix structure is called Gylase.
- Chromosome is bound to the cyplasmic membrane at one point.
- Plasmids are dispersed in cytoplasm, ring like double stranded DNA.
- Capsule, flagella
Non-essential cell components of bacteria:
Capsule:
- Outermost cover of bacteria, can be thick or thin. The host can generally not degradate the capsule and therefor it is masking the bacteria.
- Glycocalyx, slime layer
- Genotype – phenotype: Capsule has to be encoded by the bacterias genotype, but can only be expressed if the environmental factors are right, eg CO2. Therefor the environment decides the phenotype.
- Material: Polysaccharide, hyaluronic acid, polypeptide, in order of commonness. The hyaluronic acid is a common molecule of the host body and is therefor not recognized as a foreign body.
- Function: Protection and adhesion.
Example: Capsule of B.anthracis is composed of D-glutamic acid which cannot be metabolized by mammals.
- The capsulated bacteria are generally more virulent.
Flagellum (flagella):
- 15-20 mm long, diameter 10-20nm.
- Protein, flagellin(Ag), contractable.
- Threads, hook, basal body. In gram + the basal body is fixed to cytoplasmic membrane. In Gram – there are two basal bodies, one in cytoplasm and one attached to membrane.
- Flagella can be stained, but its easier to detect its presence by culturing bacteria in semisolid medium to detect movement of bacteria.
- Moving,
- Swarming, NaN3 prevent swarming.
- Important antigen.
- Fimbria and spore of bacteria.
Fimbria (pilus):
- On surface of bacteria cell. Important in attachment to target cells, especially in mucous memb. of the host. In places where there is constant movement (GIT), airflow (RT) or mucous secretion, the fimbria helps the bacteria to stay attached and don’t get washed away. It can also have a function in extending the surface of the bacteria.
- Non contractable.
- Protein, 4-10nm, 0,2-1,5 mm
- Types:
*Common fimbriae (adhesion).
*Sex fimbria (conjugation), more rigid, bacteria can attach to other bacteria and pass genetic info from one another. Only one way transport. Dangerous possibility in case of AB resistance. Can occur bw. bacterias of same spp, family and genus.
Spore of bacteria:
- Spore formation in Bacillus and Clostridia.
Endospores:
- Spores are in dormant form, they help in survival and are produced when environmental conditions are unfavorable. Spores are unique features of bacteria. In case the conditions get better again (eg water) the bacteria undergo formation to a vegetative bacteria again.
- The spore is formed when the cytoplasmic membrane invaginates and surrounds the chromosome cytoplasmic membrane around the spore. Later, another cytoplasmic membrane develops on top of the cortex.
- The genes of spore formation is scattered in the genetic material.
- Layers of the spore from inside out:
1. Inner cytoplasmic membrane
2. Cortex (cell wall)
3. Outer cytoplasmic membrane
4. Spore coat of Chitin (protein)
5. Lipoprotein membrane.
- Examination of native and stained bacteria.
Morphological examinations:
- The cell wall defines the size and shape of bacteria.
- Unstained live bacteria can be examined in two ways: wet chamber or as a hanging drop. In that way you can examine the size, shape, arrangement and movement of bacteria.
- The bacterial cells have no cell-organelles (except for mitochondria) and no contrast in the cell-material. They are therefore transparent and cannot be examined unstained in a light microscope.
Can be visualized by:
- Smear
- Staining, which can be simple or differential.
- Dyes
- Simple and differential staining methods.
- Simple staining: Simple stains will react with all microbes in an identical fashion.
-Useful solely for incr. contrast so that morphology, size, and arrangement of organisms can be determined. - Differential staining: differential stains give varying results depending on the organism being treated.
a) Gram (general differential staining)
b) Ziehl-Neelsen (acid- and alcohol fast)
c) Köster (Brucella)
d) Stamp (Chlamydia-Chlamydophila)
a) Gram staining:
1.Mix bacteria w. NaCl and spread it on the slide leaving 1 cm at each end. Fixation of the bacteria to the slide by letting it through the flame of the Bunsen burner.
2.Crystal violet: 3-5 minutes, tilt it off
3.Water - Lugol solution: 1-1.5 minutes, tilt it off. Mordant.
- 96% alcohol: 5-6 drops (alcohol extracts) Decolorization.
- Water
- Fuchsin: 0,5-1 minutes. Counterstaining.
- Water,
- Drying
Results of Gram staining:
-Crystal Violet: G+: blue, G-: blue
-Lugol solution: G+: blue, G-: blue
-Alcohol: G+: blue, G-: colourless
Fuchin: G+: blue, G-: red
b) Ziehl-Neelsen staining: - Strong carbol fuchsin (phenol): 10 min. Heat 3x until steam (to dissolve waxes): ZN+: Red, ZN-: Red
- Wash
- Sulphuric acid 5% and alcohol 96%: Several drops: ZN+: Red, ZN-: Colorless
- Wash
- Counterstain with methyleneblue: 1-2 min. ZN+: Red, ZN-: Blue
- Wash
- Bacterial metabolism.
-Imp. to know the demands of bacteria in order to propagate them because several bacteria can be differentiated according to metabolism and end products = biological examination.
-Components of bacteria: 75-80 % water, 2-15 % minerals, 2-15 % proteins (50 % carbs, generally higher in Gram-, lower in Gram+), 2-40 % lipids, wax
- Metabolism: bacteria are able to produce everything they need.
*Catabolic processes
*Anabolic processes
*Watery phase – All processes occur in this phase.
Nutrient demand:
- Carbon source + Nitrogen source - Most important demands.
- Phosphorus-, sulphur-, mineral demand
- (vitamin and additive demand)
Metabolic enzymes:
- Bacteria can only utilize small molecules, so in order to ingest large molecules the bacteria prod. EC enzymes which are excreted outside the cell to cut large molecules into smaller, ingestible molecules.
- Intra cellular enzymes
- Extra cellular enzymes
Movement of nutrients into the cell:
- Through cell wall, cytoplasma membrane
- Porins – protein tubes which can make larger components get into the cell. (Present in Gram-)
- Permeases – enzymes in the cell wall which help transport of nutrients.
- Autotrophic and heterotrophic bacteria. Vitamin and additive requirements of bacteria. Bacterial pigments.
AUTOTROPHIC bacteria:
-Utilize inorganic C and N and are absolutely saprophytes.
a) Photoautotrophic bacteria:
- Resemble plants by containing chloroplasts which are able to absord E of light and transform it to material. Difference from plants is the H-donor.
- H-donor: H2S, H2, organic metabolites (low redoxpotencial).
- Found in the environment and in the upper layer of water, both salt and fresh water.
b) Chemoautotrophic bacteria:
- E from oxidation of inorganic materials.
- Nitrification bacteria (Nitrosomonas, Nitrobacter sp.): make N usable for plants.
- Imp. in the deeper layer of waters.
HETEROTROPHIC bacteria:
- Majority are saprophytes but all pathogenic bacteria are heterotrophic.
- Organic C is needed
- N demand: inorganic/amino acids/proteins
- Some need vitamins, additives
- Paratrophic bacteria, cannot be cultured on artificial media. Need unknown additives.
- Some have CO2 demand, its needed for some pathways, eg. capsule production in B. anthracis.
Lactobacillus – in bw. hetero- and autotroph.
- Can prod. all cell components but E comes from organic C-sources.
Vitamin and additive requirement of bacteria:
Vitamin:
- Some bacteria need vitamins of group B
- Production of vitamins
Demand on additives:
- NAD (Haemophilus, Actinobacillus), V-factor
- Haem (Haemophilus), X-factor
- Mycobactin (M. avium subsp. paratuberculosis)
- DNA-hydrolisate (Mycoplasma)
Bacterial pigments:
- Pigments are secondary metabolites
- Material: Carotenoid, Phenazine
- Protection from light
- Redox processes
- Environmental effects on production of pigments
- Utilization of bacterial activity.
- Decomposition and mineralisation .Important in C, N, S cycles.
- In the handling of dung. Under anaerobic conditions and heat N→NO2→NO3.
- Sewage water (aerobic - anaerobic).
- Self cleaning of natural water.
- Production of ensilage (L. lactis, L. delbrückii, L. plantarum, anaerobic, lactic acid production, heat).
- Pickling of food, pickled cabbage, green olives.
- Production of dairy products such as yoghurt, acidophilus milk, Bulgarian milk, cultured buttermilk, cultured sour milk.
- In the fermentation industry: prod. of acids (acetic acid, lactic acid), amino acids and vitamins.
- Production of enzymes.
- Production of hormones (STH, insulin).
- Antibiotic production.
- Production of biogas.
- Microbial insecticides (B. thuringiensis).
- Paint digesting.
- Bioremediation: inactivation of natural oil, diesel oil, wood conservation materials.
- Biomining.
- Desulphurization of coal (bränsle).
- Production of artificial snow (P. syringae)
- Nitrogen metabolism of bacteria.
Nitrogen-sources:
- Protein to produce aa, generally not essentiall (Arcanobacterium, dermatophillus).
- Amino acid
- Other organic nitrogen compounds
- Some heterogenous bacteria can utilize ammonium salts, ammonia.
- Mainly autotrophic bacteria utilize N2.
Nitrogen demand of bacteria:
- Protein demand
- Amino acid demand of fastidious bacteria.
- Inorganic nitrogen demand
1. Proteolytic bacteria:
- Use proteolytic enzymes to gain aa and oligo peptides:
Aerobic and anaerobic proteolytic bacteria
- Products: aa and end products of aa.
- Detection of the most important enzymes and metabolites of the nitrogen metabolism.
There are two main ways of utilizing aa by bacteria:
1) Decarboxylation
2) Deamination
Products:
- Amines: cadaverine, putrescine, histamine, toxic to for the living cells.
- Keto-acids
- Acetic acid, lactic acid, butyric acid, valeric acid
- NH3, H2, CO2
- CO2, H2O
- Other: H2S, indole, NH3
Test for detecting urease activity:
- Prod. of urease enzyme is tested with a medium containg urea. Inoculate bacteria and wait for colour change as a pH indicator of enzyme activity:
- Uninoculated tube: orange, pH 7,2
- Negative test: yellow, acidic.
- Positive test: Pink, alkaline.
- Carbohydrate metabolism of heterotrophic bacteria.
Carbohydrate metabolism, energy production:
1. Oxidation production: in presence of O2.
2. Fermentation
(Two most important E sources. Occurs in cytoplasmic membrane of bacteria. From carbs.)
3. Decarboxylation - Deamination
4. Hydrolysis
Oxygen demand:
- Obligate aerobic, Bacillus, micrococcus, microbacteria.
- Obligate anaerobic, Clostridia
- Facultative anaerobic (aerobic facultative anaerobic)
1) Fermentation:
- Breakdown of carbs in absence of O2.
- Oxidation w. dehydrogenation.
- Can utilize a wide range of nutrients: Polysaccharids, oligosaccharids, disaccharids, monosaccharids, alcohols, glycosides etc.
- Mainly: basic Glu, F-6-P (through GL) and prod. piruvic acid (lactic acid + NADH+H+: H→piruvic acid, organic acids, aldehydes, cystine, thioglycolate, thiosulfate).
-If there is excess of carbs: lactic acid is prod. (major end prod.).
-If there is limited amount of carbs: organic acids, alcohols, gas is prod.
-From aa → organic acids (keto-acids), NH3, CO2 can be prod.
-Fast way of gaining E but not very efficient.
2) Oxidation:
- Occurs in presence of O2.
- Begins with dehydrogenation (same step as in fermentation).
- Enters TCA cycle (E prod)
- NADH+H+ (H activated) → flavin enzymes (flavin-mononucleotid, flavin-adenin-dinucleotid) → H+
- When the electron is passed to N the bacteria are called nitrification bacteria. They´re very imp. for eco-system: make N utilizable for the plants.
- Electron→cytochrom-system→O2- or NO2, NO3, SO4 (denitrification bacteria)
- H2O2 and E are produced, H2O2 → H2O and CO2
