1. MLAB 2434 – CLINICAL MICROBIOLOGY SUMMER, 2005 CECILE SANDERS & KERI BROPHY

2. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics Taxonomy Kingdom, Division, Class, Order, Family, Tribe, Genus and Species Family = “Clan”; has “–aceae” ending Genus = “Human last name” Species = “Human first name” When in print, genus and species are italicized. (Staphylococcus aureus) When written, genus and species are underlined. (Staphylococcus aureus)

3. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Staphylococcus sp. is used when referring to the genus as a whole when the species is not identified. “sp.” – singular (Staphylococcus sp.) “spp.” – plural (Staphylococcus spp.)

4. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Bacteria Identification – test each bacterial culture for a variety of metabolic characteristics and compare the results with known results. All organisms are either “prokaryotes”, “eukaryotes”, or “archaeobacteria”

5. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) EUKARYOTES - fungi, algae, protozoa, animal cells, and plant cells Cells have nuclei that contains DNA and are complex Most cells do NOT have a cell wall

6. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) PROKARYOTES - bacteria Do not have a membrane-bound nucleus DNA is a single circular chromosome Have both cell (plasma) membrane AND cell wall.

7. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Archaeobacteria Resembles eukaryotes Found in microorganisms that grow under extreme environmental conditions See chart on page 6 for comparisons of Prokaryotes and Eurkaryotes

8. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Bacterial Cell Wall

9. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Gram Positive (G+) Cell Wall Very thick protective peptidoglycan layer Many G+ antibiotics act by preventing synthesis of peptidoclycan Consists of cross-linked chains of glycan Also contain teichoic acid and liptoeichoic acid Unique structure makes these bacteria G+

10. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Gram Negative (G-) Cell Wall Two layers; inner is much thinner than G+ cell walls Outer wall contains several molecules, including Lipid A which is responsible for producing fever and shock in infections with G- bacteria

11. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) G+ cocci in clusters → G- bacilli (rods) →

12. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Acid Fast Cell Wall – mainly Mycobacteria and Nocardia Have a G+ cell wall structure but also a waxy layer of glycolipids and fatty acids (mycolic acid) Waxy layer makes them difficult to gram stain Can be decolorized by acid-alcohol, hence the name “acid fast”

13. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Absence of Cell Wall – mainly Mycoplasma and Ureaplasma Lack of cell wall results in a variety of shapes microscopically Some bacteria produce slime layers

14. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Some bacteria produce a capsule Protect the bacteria from phagocytosis Capsule usually does not stain, but can appear as a clear area (halo-like)

15. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Cell Appendages Flagella – exterior protein filaments that rotate and cause bacteria to be motile Polar Peritrichous Pili (fimbriae) – hairlike projections that aid in attachment to surfaces

16. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Microscopic Shapes Cocci (spherical) Bacilli (rod-shaped) Spirochetes (helical) Groupings Singly Pairs Clusters Chains Palisading

17. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Size and length Short Long Filamentous Fusiform Curved Pleomorphic

18. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)

19. Common Bacterial Stain Gram Stain – to be covered in lab Acid-fast

20. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Acridine Orange – stains nucleic acid of both G+ and G- bacteria, either living or dead; used to locate bacteria in blood cultures and other specimens where background material obscures gram stains

21. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Methylene Blue – stain for Corynebacterium diphtheriae to show metachromatic granules and as counter-stain in acid-fast stain procedures Lactophenol Cotton Blue – fungal stain Calcofluor White – fungal stain

22. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) India Ink – negative stain for capsules

23. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Microbial Growth and Nutrition Needs Source of carbon for making cellular constituents Source of nitrogen for making proteins Source of energy (ATP) for cellular functions Smaller amounts of other molecules

24. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Nutritional Requirements for Growth Autotrophs (lithotrophs) Able to grow simply, using only CO 2, water and inorganic salts Obtain energy via photosynthesis or oxidation of inorganic compounds Occur in nature and do not normally cause disease

25. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Heterotrophic Require more complex substances for growth Require an organic source of carbon and obtain energy by oxidizing or fermenting organic substances All human bacteria fall in this category Within this group, nutritional needs vary greatly

26. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Types of Growth Media Minimal medium – simple; not usually used in diagnostic clinical microbiology Nutrient medium – made of extracts of meat or soy beans Enriched medium – nutrient medium with extra growth factors, such as blood

27. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Selective medium – contains additives that inhibit the growth of some bacteria while allowing others to grow Differential medium – contains additives that allow visualization of metabolic differences in bacteria Transport medium – holding medium to preserve those bacteria present but does not allow multiplication

28. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Environmental Factors Influencing Growth pH – most media is between 7.0 and 7.5 Temperature – most pathogens grow at body temperature; grown at 35° C in the lab

29. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Gaseous composition Obligate aerobes – require oxygen Obligate anaerobes – cannot grow in the presence of oxygen Facultative anaerobes – can grow with or without oxygen Capnophilic – grow better with extra CO 2

30. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Bacterial Growth Reproduce by binary fission Can be fast (as little as 20 minutes for E. coli) or slow (as long as 24 hours for M. tuberculosis) Determination of Numbers Direct counting under microscope Direct plate count Density measurement

31. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Bacterial Biochemistry and Metabolism Metabolic reactions cause production of energy in form of ATP Identification systems analyze unknown specimens for: Utilization of variety of substances as a source of carbon Production of specific end products from various substrates Production of acid or alkaline pH in the test medium

32. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Fermentation and Respiration (Oxidation) Fermentation Anaerobic process in obligate and facultative anaerobes The electron acceptor is an organic compound Does NOT require oxygen

33. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Oxidation More efficient energy-generating process Molecular oxygen is the final electron acceptor Aerobic process in obligate aerobes and facultative anaerobes

34. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Metabolic Pathways – main one is Embden- Meyerhoff Convert glucose to pyruvic acid, a key intermediate

35. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) From pyruvic acid: Alcoholic fermentation (ethanol) Homolactic acid fermentation (lactic acid) Heterolactic acid fermentation (lactic acid, CO 2, alcohols, formic and acetic acids Propionic acid Mixed acid fermentation (lactic, acetic, succinic, and formic) Butanediol fermentation Butyric acid fermentation

36. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Main oxidative pathway is the Krebs Cycle, resulting in acid and CO 2 Carbohydrate Utilization & Lactose Fermentation “Sugars” = carbohydrates Lactose fermentation – key component in identification schemes Lactose is converted to glucose, so ALL lactose fermenters also ferment glucose

37. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Genetic Elements and Alterations Plasmid Extra piece of DNA Code for antibiotic resistance and other virulence factors are often found on plasmids Sometimes passed from one bacterial species to another

38. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Plasmid replication

39. Chapter 1 - Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Mutations Changes that occur in the DNA code Results in changes in the coded protein or in the prevention of its synthesis