Institute for Microbiology, Medical Faculty of Masaryk University and St. Anna Faculty Hospital in Brno Miroslav Votava BACTERIAL GROWTH The 3rd lecture for 2nd-year students of General Medicine March 2, 2015 Size of bacteria – revision •Pathogenic bacteria: mainly around 1 – 5 μm (1 μm = 10-3 mm) •Genus Staphylococcus: the diameter circa 1 μm •Relatively big: genera Bacillus and Clostridium (robust rods around 1 – 2 × 10 μm) •Relatively long: old cultures of most rods • (fibers up to 50 μm long) •Relatively small: genus Haemophilus (in the sputum approximately 0.3 × 0.6 μm) •Even smaller: rickettsiae (circa 0.5 μm) chlamydiae (elementary bodies circa 0.3 μm) mycoplasmas (circa 0.2 – 0.25 μm ) Arrangement and shape of cocci – revision a)in clumps: Staphylococcus aureus b)in chains: Streptococcus pyogenes c)lancet-like diplococci: Streptococcus pneumoniae d)flattened diplococci: Neisseria gonorrhoeae e)cocci in tetrads: Micrococcus luteus f) obr_2a Arrangement and shape of rods I –revision a)absolute majority of rods: e.g. Escherichia coli b)delicate streptobacilli: Haemophilus ducreyi c)coccobacilli in pairs or diplobacilli: Moraxella lacunata d)robust rods, rounded ends: Clostridium perfringens e)robust rods, flat up to concave ends, bamboo cane-like chains: Bacillus anthracis obr_2b Arrangement and shape of rods II – revision • f) club-like in palisades: Corynebacterium diphtheriae •g) slender, in hinted palisades: Mycobacterium tuberculosis •h) branched, fragmented: Nocardia asteroides • i) spindle-like: Fusobacterium fusiforme • j) minute, pleomorphic: Haemophilus influenzae • obr_2b Curved and spiral rods – revision •a) curved rods, crescent-shaped: Vibrio cholerae •b) thick spirals: Spirillum minus •c) uneven spirals: Borrelia recurrentis •d) delicate, regular spirals: Treponema pallidum •e) very fine spirals with bent ends: Leptospira icterohaemorrhagiae • obr_2c Outline of bacterial cell – revision capsule cytoplasmic membrane bacterial cell wall fimbriae nucleoid ribosomes plasmids granula vacuole flagellum Bacterial cell wall – revision •G+ G– • lipoteichoic acid O-antigen lipopoly- • inner polysaccharide saccharide • lipid A (endotoxin) • murein • • • porin • outer • membrane • lipoprotein • periplasmatic • space • • innner membrane •cytoplasmic membrane (G+) (G–) Gram staining – revision • G+ G– 1.Fixation by flame 3 times 2.Gram stain 20 s 3.Lugol solution 20 s 4.Alcohol max. 20 s 5.Aqua fontis rinse 6.Safranin 1 min 7.Aqua fontis rinse 8.Drying Sensitivity to antibiotics – revision •Effect mostly •on Gram-positives: • •beta-lactams (penicillin, • oxacillin = methicillin) •macrolides (erytromycin) •lincosamides (lincomycin) •glycopeptides (vancomycin) • • • •Effect mostly •on Gram-negatives: • •aminoglycosides (gentamicin) •monobactams (aztreonam) •polypeptides (colistin) •3rd gen. cephalosporins • (cephtriaxon) Resistance to the environment – an addition •Gram-positives • •They endure well drying up and higher salt concentrations •→ and so we find them: •on skin (staphs, propionibacteria) •in soil (clostridia, bacilli, nocardiae, moulds) • •Gram-negatives • •They endure well the effect of toxic substances and extremes of pH •→ and so we find them: •above all in moist places (enterobacteriae, pseudomonads, other non-fermenting rods, vibria) Growth cycle of bacteria •Bacteria reproduce by binary fission •Period I (initiation): the cell grows, inside it proteins initiating the next step accumulate •Period C (chromosome replication): the chromosome diverges from one spot in both directions opposite one another •Period D (division): –supply of macromolecules is created –cytoplasmatic membrane inserts between the replicated chromosomes and separates them –cell wall grows into the cell at a particular spot and forms a septum that ultimately divides the maternal cell into two daughter cells Division of bacterial cell capsule cytoplasmatic membrane bacterial cell wall fimbriae nucleoid ribosomes plasmids granules vacuole flagellum septum Division & arrangement of cocci •Cocci, dividing in one plane: streptococci chains • •Cocci, in different planes: staphylococci clumps • • •Cocci, in two perpendicular planes: • micrococci tetrads • • •Notice that after dividing cocci touch each other! Division and arrangement of rods •Rods, transverse division: majority (chains of • rods) • • •Rods, lengthwise division: mycobacteria • corynebacteria • (arrangement in palisades) Generation time •Generation time = duration of the growth cycle = = duplication time = duration of doubling the number of bacteria •Generation time of bacteria: on average cca 30 min •Escherichia coli • under ideal conditions 20 min •Mycobacterium tuberculosis • approximately 12 hrs •Since during each generation time the number of bacteria doubles, bacteria multiply by geometric progression Geometric progression – I •Number of bacteria by generation time 0.5 hour • time (hrs) number time (hrs) number 0 20=1 4 28=256 0.5 21=2 4.5 29=512 1 22=4 5 210=1024 1.5 23=8 5.5 211=2048 2 24=16 6 212=4096 2.5 25=32 12 224 ≈ 107 3 26=64 18 236 ≈ 1011 3.5 27=128 24 248 ≈ 1014 Geometric progression – II •If the generation time is 30 min, after 24 hrs theoretically one cell gives origin to 248 = 2.8×1014 cells, actually it is by approximately 5 orders less (i.e. around 109 cells) •109 bacteria is such an amount that it is visible even by the naked eye: •Liquid medium (broth) becomes 1. cloudy or 2. a sediment appears at the bottom or 3. a pellicle is seen at the top •On a solid medium (agar) a bacterial colony forms • • What is a bacterial colony? •Bacterial colony = a form on the surface of the agar, containing mutually touching cells, cca 109 living and cca 105 already dead • •Appearance of the colony depends apart from other things on the • 1. microbial species (e.g. on the size of its cells) • 2. sort of culture medium (e.g. on the amount of its nutrients) • 3. distance among colonies (the higher distance, the larger and more typical the colony) • • Features of a bacterial colony •Bacterial colony can have up to 10 features: • 1. Size – usually around 1-2 mm • 2. Shape – round, oval, irregular, lobular etc. • 3. Profile – flat, convex, dish-shaped etc. • 4. Margins – straight, fibrous, with projections etc. • 5. Surface – smooth & glossy, matt, rough, wrinkled • 6. Transparency – transparent, nontransparent • 7. Colour – colourless, pigmented (yellowish etc.) • 8. Changes in vicinity – pigmentation, haemolysis • 9. Consistency – sticky, mucous, crumbly, rooted •10. Smell – foul, pungent, of jasmin, sperm, fruit etc. • Geometric progression – III •Consequences will become evident by the quantitative examination of urine: •From the external orifice of urethra bacteria can be flushed into urine up to the concentration of 103/ml •= a mere contamination (in cystitis the urine contains >105 bacteria/ml, i.e. >105 CFU, colony forming units) •In 1 μl of this urine there will be 1 bacterium (1 CFU) •→ in this case from 1 μl only 1 colony will appear •The result of the examination will be: • 103 CFU/ml = probably contamination •However, it applies only when the urine is processed immediately •But what if the urine takes several hours to get into the laboratory in the hot summer? Geometric progression – IV •Urine is a good culture medium, bacteria multiply in it even during the transportation •At the generation time of 30 min: •After 2 hrs: from 1000 cells → 16,000 cells from 1 μl of urine 16 colonies will grow •The result: • 104 CFU/ml = suspect finding •After 4 hrs: from 1000 cells → 256 000 cells from 1 μl of urine 256 colonies will grow •The result: • >105 CFU/ml = positive finding (of course a false one!) •→ the urine must be processed up to 2 hrs after the sampling or placed in refrigerator at 4 °C • Microbial growth curve – I •The result 109 cells/24 hrs applies for the stationary culture, in which nutrients are consumed and products of metabolism accumulate • the speed of multiplication changes depending on time • growth phases exist that can be depicted by the growth curve • Microbial growth curve – II •Growth curve depicts the number of viable cells in the logarithmic scale, depending on the age of culture •Growth phases 1.lag phase 2.log (exponential) phase 3.stationary phase 4.death phase •There are gradual transitions between the phases Microbial growth curve – III 10 8 6 4 2 lag phase stationary phase approximately 24 hrs time What is a logarithm? •In the equation •103 = 1000 •10 is a base, 3 is an exponent •The exponent (3) = logarithm of the number 1000 (at the base 10) •Logarithms at the base 10 = common logarithms • •In general: •Logarithm of the number a is an exponent (e) to the power of which the base (B) is raised so that it equals the number a •Therefore: if a = Be, then logB a = e •Example: if a = 1000 = 103 (and B = 10), then log a = 3 • • Microbial growth curve – III 10 8 6 4 2 lag phase stationary phase approximately 24 hrs time Microbial growth curve – IV Continuous culture Recommended reading material •Paul de Kruif: Microbe Hunters •Paul de Kruif: Men against Death •Axel Munthe: The Story of San Michele • • •mvotava@med.muni.cz • •Thank you for your attention • •