1. Unit 1 Chapter 1 Bacterial Cell Structure CLS 3303 Clinical Microbiology

2. Taxonomy Defined as the orderly classification & grouping of organisms into categories Kingdom, Division, Class, Order, Family, Tribe, Genus and Species ( these are the formal levels of classification)  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) To abbreviate organism names: use first letter capitalized of the genus followed by a period and the species epithet. ( i.e S. aureus)

3. Nomenclature 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. Classification by Cellular Type 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. Classification by Cellular Type: Prokaryotes PROKARYOTES - bacteria Do not have a membrane- bound nucleus DNA is a single circular chromosome and RNA are free in the cytoplasm Have both cell (plasma) membrane AND cell wall. Have no mitochondria, endoplasmic reticulum (ER) or Golgi bodies

6. Classification by Cellular Type: Eukaryotes 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 (Fungi have cell walls made chitin)

7. Classification by Cellular Type: Archaeobacteria Resembles eukaryotes Found in microorganisms that grow under extreme environmental conditions Cell wall lacks peptidoglycan See chart on page 5 for comparisons of Prokaryotes and Eurkaryotes

8. Prokaryotic & Eukaryotic Cell Comparison

9. Bacterial Cell Wall

10. Gram Positive (G+) Cell Wall Very thick protective peptidoglycan layer Many G+ antibiotics act by preventing synthesis of peptidoglycan Consists of cross-linked chains of glycan Also contain teichoic acid and lipoteichoic acid Unique structure makes these bacteria G+ by protecting against the decolorizing step in Gram staining

11. Gram Negative (G-) Cell Wall Two layers; outer 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 The thin walls allow the decolorizer to enter the cell and take out the crystal violet stain.

12. (G+) and (G-) Microorganisms G+ cocci in clusters→ G- bacilli (rods)→ When identifying bacteria, remember that rods can sometimes be short and look like cocci, but cocci do not look like rods

13. 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 ). It is hydrophobic and affects permeability Waxy layer makes them difficult to gram stain (More than 60% of the cell wall is lipid) Cannot be decolorized by acid-alcohol, hence the name “acid fast” Bacteria is pink Background is green or blue

14. Absence of Cell Wall Mainly Mycoplasma and Ureaplasma Lack of cell wall results in a variety of shapes microscopically Contain sterols in cell membrane

15. Surface Polymers: Slime Layers Some bacteria produce slime layers Made of polysaccharides Inhibit phagocytosis and also help to attach to the host.

16. Surface Polymers: Capsule Some bacteria produce a capsule Protect the bacteria from phagocytosis Capsule usually does not stain, but can appear as a clear area (halo-like)

17. Cell Appendages Flagella – exterior protein filaments that rotate and cause bacteria to be motile  Polar –Extend from one end –Can occur singly or in multiple tufts  Peritrichous –Flagella found on all sides of bacteria Pili (fimbriae) – hairlike projections that aid in attachment to surfaces

18. Examples of Flagella

19. Bacterial Morphology Microscopic Shapes Cocci (spherical) Bacilli (rod-shaped) Spirochetes (helical) Groupings Singly Pairs Clusters Chains Palisading

20. Bacterial Morphology (cont’d) Size and length Short Long Filamentous: Fusiform: bacilli with tapered, pointed ends Curved Pleomorphic: variance in size & shape within a pure culture

21. Other Common Bacterial Stains: Acridine Orange (fluorochrome dye) 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

22. Other Common Bacterial Stains: Methylene Blue Stain for Corynebacterium diphtheriae to show metachromatic granules and as counter-stain in acid-fast stain procedures

23. Other Common Bacterial Stains: Lactophenol Cotton Blue – fungal stain

24. Other Common Bacterial Stains: Calcuflour White – fungal stain A fluorochrome that binds to chitin in fungal cell walls Apple-green or blue- white with a fluorescent microscope

25. Other Common Bacterial Stains: India Ink Negative stain for capsules, surrounds certain yeasts

26. Other Common Bacterial Stains: Endospore stain Heat is used to help the primary stain (Malachite green) into the spore. The spore stains green The counter stain, (safranin) stains the rest of the organism

27. 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

28. 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

29. Nutritional Requirements for Growth 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

30. 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 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

31. 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

32. Environmental Factors Influencing Growth 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 (5 -10%)  Microaerophilic- grow better in low oxygen environments ( about 20%)  Campylobacter spp. require 5 – 6% oxygen

33. 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 )

34. Determination of the Number of bacterial cells Direct counting under microscope: estimate the number of bacteria in a specimen. Does not distinguish live or dead cells Direct plate count: grown from dilutions of broth cultures. Counts viable cells only. Colony Forming Units (CFU/mL) Density measurement: (turbidity) bacterial broth culture in log phase

35. 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

36. Fermentation  Anaerobic process in obligate and facultative anaerobes  The electron acceptor is an organic compound  Does NOT require oxygen

37. Oxidation (Respiration)  More efficient energy-generating process  Molecular oxygen is the final electron acceptor  Aerobic process in obligate aerobes and facultative anaerobes

38. Metabolic Pathways Main one is Embden-Meyerhoff Convert glucose to pyruvic acid, a key intermediate Generates energy in the form of ATP

39. Metabolic Pathways From pyruvic acid: Alcoholic fermentation (ethanol) (ex: yeast) Homolactic acid fermentation (lactic acid) )ex: strep) Heterolactic acid fermentation (lactic acid, CO 2, alcohols, formic and acetic acids Propionic acid Mixed acid fermentation (lactic, acetic, succinic, and formic) (ex: e-coli and salmonells) Butanediol fermentation: (ex: Kleb, enterobacter & serratia) Butyric acid fermentation: (ex: obligate anaerobe)

40. Metabolic Pathways 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

41. 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. This is how resistance is acquired.

42. Plasmid Replication

43. Genetic Elements and Alterations Mutations “They don’t always read the book” Changes that occur in the DNA code Results in changes in the coded protein or in the prevention of its synthesis

44. References http://media.photobucket.com/image/micro/lovite x2000/Micro%20biology%20lab/b1cf.jpg?o=81 http://media.photobucket.com/image/micro/lovite x2000/Micro%20biology%20lab/b1cf.jpg?o=81 http://nhscience.lonestar.edu/biol/wellmeyer/bact eria/capsules3.jpg http://nhscience.lonestar.edu/biol/wellmeyer/bact eria/capsules3.jpg http://www.iccb.state.il.us/pt3/images/sci/mod11/ bacillus_subtilis.jpg http://www.iccb.state.il.us/pt3/images/sci/mod11/ bacillus_subtilis.jpg