1. Institute for Microbiology, Medical Faculty of Masaryk University and St. Anna Faculty Hospital in Brno Miroslav Votava MORPHOLOGY AND STRUCTURE OF BACTERIAL CELL The 2nd lecture for 2nd-year students February 24, 2014

2. Objects of the Medical Microbiology – revision a)Pathogenic microbes (causing diseases of human beings or animals) b)Normal microflora (microbes commonly present in healthy persons or animals) c)Mutual relationship between microbes and their hosts (how we defend themselves against the microbes or how we utilize their presence) d)Relationship between microbes and the environment (including methods how to eradicate the microbes)

3. Different objects and sections of microbiology – revision bacteriabacteriologybacteriabacteriology micromycetes (mouldsmicromycetes (moulds & yeasts) mycology (algae) ( algology)(algae) ( algology) parasites parasitologyparasites parasitology –protozoaprotozoology –helminthshelminthology –arthropodsentomology virusesvirologyvirusesvirology General microbiology × special microbiology

4. “Must-knows” about microbes for an E mark – revision Pathogenicity 1. Which diseases or syndromes does the microbe in question cause? 2. How are they called in Latin? Etiology 3. Which microbe is the etiological agent of the infectious disease in question? 4. Which microbes (bacteriae, yeasts, moulds, viruses or parasites) are the most important causes of the syndrome in question? Treatment 5. What is the treatment of choice? 6. If it is an antibotics, which one?

5. “Must-knows” for an A mark I – revision 1. Classification, morphology, structure Is it a bacterium, yeast, mold or protozoan? If a bacterium: is it Gram-positive, Gram-negative, or does it stain in yet another way? Is it a coccus, rod, filament, spiral? How are the cells relatively arranged? In pairs, clumps, chains, tetrads? Do they produce spores, capsules, granules? 2. Physiology, biochemistry, genetics, tenacity Is it a strict anaerobe (does the oxygen kill it)? Has it any important bichemical property? Or genetical one? Is it resistant (robust), or delicate?

6. “Must-knows” for an A mark II – revision 3. Antigenic structure Does it exist in one antigenic type or in several ones? 4. Pathogenicity Which diseases or syndromes does it cause? How are they called in Latin? 5. Pathogenesis Portal of entry, spread through the body, elimination? How do the symptoms develop? Which factors of pathogenicity (virulence) has it? 6. Immunity Does it actually develop after the contact with the microbe? Is it short-lasted, or life-long; humoral, or cellular one?

7. “Must-knows” for an A mark III – revision 7. Epidemiology What is the source of infection: man, animal or environment? How is the agent transmitted? 8. Prevention, if necessary prophylaxis Does a vaccination exist, or passive immunization? What type of vaccine is in the use? 9. Treatment What is the treatment of choice? If it is an antibotics, which one?

8. “Must-knows” for an A mark IV – revision 10. Laboratory diagnostics Direct demonstration (= detection of the agent in question – microscopy, culture, detection of antigens, nucleic acid), or indirect one (= detection of antibodies)? indirect one (= detection of antibodies)? If direct one, what is sampled from the patient? In which way is the specimen examined? If by the culture, is a special medium required? How is the isolated strain identified? If indirect one, is a special serologic reaction required? What is considered as a positive result?

9. Shape of bacteria – I Spherical shape: cocci regular sphere: staphylococci flattened: gonococci, meningococci pointed (lancet-like): pneumococci Elongated shape: rods straight: majority (e.g. Escherichia coli) short (coccobacilli): acinetobacters long (fibers): mainly old cultures slender: Mycobacterium tuberculosis robust: majority of bacilli, lactobacilli, Clostridium perfringens (to be continued)

10. Shape of bacteria – II (rods, continued:) with split ends: bifidobacteria branched: nocardiae, actinomycetes curved: vibria, campylobacters with flat ends: Bacillus anthracis spindle-shaped: fusobacteria club-shaped: corynebacteria pleomorphic: haemophili

11. Shape of bacteria – III Spirals: thick: Spirillum uneven: Borrelia regular: Treponema slender with bend ends: Leptospira

12. Size of bacteria 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 )

13. Arrangement and shape of cocci 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

14. Arrangement and shape of rods – I 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

15. Arrangement and shape of rods – II f) club-like in palisades: Corynebacterium diphtheriae f) club-like in palisades: Corynebacterium diphtheriae g) slender, in hinted palisades: Mycobacterium tuberculosis h) branched, fragmented: Nocardia asteroides i) spindle-like: Fusobacterium fusiforme i) spindle-like: Fusobacterium fusiforme j) minute, pleomorphic: Haemophilus influenzae j) minute, pleomorphic: Haemophilus influenzae

16. Curved and spiral rods 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

17. Outline of bacterial cell capsule cytoplasmic membrane bacterial cell wall fimbriae nucleoid ribosomes plasmids granula vacuole flagellum

18. Bacterial cell wall G+ G– lipoteichoic acid O-antigen lipopoly- lipoteichoic acid O-antigen lipopoly- inner polysaccharide saccharide inner polysaccharide saccharide lipid A (endotoxin) lipid A (endotoxin) murein murein porin porin outer outer membrane membrane lipoprotein lipoprotein periplasmatic periplasmatic space space innner membrane innner membrane cytoplasmic membrane (G+) (G–)

19. Gram staining Gram staining G+ G– 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

20. Basis of Gram-positiveness Rather a puzzle – but it is connected with the structure of cell wall The 1st theory: Thick peptidoglycane (murein) layer contracts after the alcohol and slows down the washing of crystal violet and iodine complex out of Gram-positive cells The 2nd theory: Cell wall of Gram-negative bacteria contains more lipids, therefore the alcohol forms pores in the wall and the colored complex can be washed out easier

21. Examples of G+ and G– microbes Gram-positivesStaphylococcusStreptococcusBacillusLactobacillusClostridiumListeriaCorynebacterium Yeasts and moulds Gram-negativesEscherichiaSalmonellaVibrioHaemophilusPseudomonas All spirals Mycoplasma Rickettsiae & chlamydiae

22. Sensitivity to antibiotics Effect mostly on Gram-positives: beta-lactams (penicillin, oxacillin = methicillin) oxacillin = methicillin) macrolides (erytromycin) lincosamides (lincomycin) glycopeptides (vancomycin) Effect mostly on Gram-negatives: aminoglycosides (gentamicin) monobactams (aztreonam) polypeptides (colistin) 3rd gen. cephalosporins (cephtriaxon)

23. Recommended reading material Paul de Kruif: Microbe Hunters Paul de Kruif: Men against Death Could you kindly supply me with another work in connection with microbes or at least medicine? Please mail me your suggestions at: mvotava@med.muni.cz Thank you for your attention