1. Principles of Bacteriology Prepared by Hamad ALAssaf Alassaf_h@yahoo.com 2015 1

2. Bacterial Structure Peptidoglycan :  Peptidoglycan : gives rigid support, protect against osmotic pressure.  Capsule:  Capsule: protect against phagocytosis. Polysaccharides except Bacillus anthrax, which contains D-glutamate.  Spore:  Spore: provides resistance to dehydration, heat, and chemicals. Keratin-like coat; dipicolinic acid. 2

3. Gram Stain Limitations These organism do not Gram stain well:These organism do not Gram stain well: 1- Treponema (too thin to be visualized). 2- Rickettsia (intracellular parasite). 3- Mycobacteria (No cell wall). 4- Legionella pnumophila ( primarily intracellular). 5- Chlamydia (intracellular parasite; lacks muramic acid in cell wall). 3

4. Stains Giemsa : Giemsa : Borrelia, Plasmodium, Trypanosomes, Chlamydia. PAS (periodic acid-Schiff):PAS (periodic acid-Schiff): Stains glycogen, mucopolysaccharides; used to diagnose Whipple’s disease (Tropheryma whippelii). Ziehl-Neelsen:Ziehl-Neelsen: Acid-fast organism. Indian ink:Indian ink: Cryptococcus neoformans, and used to stain thick polysaccharide capsule red. Silver stain:Silver stain: Fungi (e.g. Pneumocystis), Legionella. 4

5. Silver Stain Indian ink Giemsa Stain ZN stain 5

6. Special culture requirements Microrganism Media used for isolation H. influenzae Chocolate agar with factors V (NAD) and X (hematin). N. gonorrhoeae Thayer-Martin Media (VPN) B. pertussis Bordet-Gengou agar C. diphtheriae Tellurite plate, Loffler’s media M. tuberculosis Lowenstein-Jensen agar M. pneumoniae Eaton’s agar Legionella Charcoal yeast extract agar buffered with cysteine Fungi Sabouraud’s agar Enteric pathogens Hektone enteric agar or Xylose-Lysine-Deoxycholate agar Vibrio cholerae TCBS (alkaline growth medium) 6

7. Encapsulated Bacteria:Encapsulated Bacteria: Positive Quellung reaction e.g. Streptoccoccus pneumonia, Klebsiella pneumonia, Haemophilus influnesae type B, Neisseria meningitides, Salmonella, Group B streptococcus. Urease-positive bugs:Urease-positive bugs: Proteus, Klebsiella, H.pylori, Ureaplasma. Pigment-producing bacteria:Pigment-producing bacteria: Actinomyces israelii (yellow –sulfur), S.aureus (yellow pigment), Pseudomonas aeruginosa (blue-green pigment), Serratia marcescens (red pigment). 7

8. Bacterial virulence factors These promote evasion of host immune response. 1- Protein A (S. aureus): 1- Protein A (S. aureus): Binds Fc region of Ig. Prevent opsonization and phagocytosis. 2- IgA protease: 2- IgA protease: Enzyme that cleaves IgA. Secreted by S.pneumoniae, H. influenza type B, and Neisseria in order to colonize respiratory mucosa. 3- M protein (group A streptococcus): 3- M protein (group A streptococcus): helps prevent phagocytosis. 8

9. Normal Flora Is found on body surfaces contiguous with the outside environment. Is semi-permanent, varying with major life changes. Can cause infection: - if misplaced, e.g. fecal flora to urinary tract or abdominal cavity, or skin flora to catheter. - if person become compromised, normal flora may overgrow (oral thrush). contribute to health: - Protective host defense by maintaining conditions such as pH so other organism may not grow. - Serves nutritional function by synthesizing: K and B12 vitamins.  Normal Flora e.g. Nose (S.aureus), cutaneous (Staphylococcus epidermidis), Oropharynx (Viridans streptococci), Vagina (Lactobacillus), colon (E.coli).  Blood and Stomach: No Normal Flora present. 9

10. Colonization The first stage of microbial infection is colonization: Pathogens usually colonize host tissues that are in contact with the external environment. Sites of entry in human hosts include the urogenital tract, the digestive tract, the respiratory tract and the conjunctiva. Adherence to cell surfaces involves: -Pili/fimbriae: primary mechanism in most gram negative cells. -Teichoic acids: primary mechanism of gram positive cells. -Adhesion: colonizing factor adhesions, pertussis toxin, and hemagglutinins. -IgA proteses: cleaved Fc portion may coat bacteria and bind them to cellular Fc receptor. 10

11. Definitions Carrier: Carrier: person colonized by a potential pathogen without overt disease. Bacteremia: Bacteremia: bacteria in bloodstream without overt clinical signs. Septicemia: Septicemia: bacteria in bloodstream (multiplying) with clinical symptoms.  Spores of fungi have a reproductive role. 11