1. MLAB 2434 – CLINICAL MICROBIOLOGY KERI BROPHY-MARTINEZ

2. Bacterial Cell Structure, Physiology, Metabolism, & Genetics
Taxonomy
Defined as the orderly classification & grouping of organisms into categories
Based on genotype and phenotype
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”

3. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Nomenclature
Family names: Capitalized with “aceae” endings
Genus names: Capitalized
When in print, genus and species are italicized. (Staphylococcus aureus)
When written, genus and species are underlined. (Staphylococcus aureus)
Species names
Staphylococcus sp. is used when referring to the genus as a whole when the species is not identified.
Can be referred to as singular or plural
“sp.” – singular (Staphylococcus sp.)
“spp.” – plural (Staphylococcus spp.)
Abbreviations
First letter of the genus, followed by a period and the species epithet
Ex: Staphylococcus aureus changes to S. aureus

4. 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. 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.
F= flagellum
C=capsule
P= pili
N= nuclear info
R=ribosome
CM= cytoplasmic membrane
CW= cytoplasmic wall

6. 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
V=Vesicle
M=Mitochondria
G= Golgi
NM= nuclear membrane
N= nucleus
NC= nucleolus
RER= rough endoplasmic reticulum
PM= plasma membrane

7. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Archaeobacteria
Resembles eukaryotes
Found in microorganisms that grow under extreme environmental conditions
Cell wall lacks peptidoglycan

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

10. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Gram Positive (GP) Cell Wall
Very thick protective peptidoglycan layer
Many GP antibiotics act by preventing synthesis of peptidoglycan
Consists of cross-linked chains of glycan
Also contain teichoic acid and lipoteichoic acid these unique structures makes these bacteria GP

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

12. The Cell Wall
Gram Stain
Gram Positive
Gram Negative

13. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
GP cocci in clusters →
GN bacilli (rods) →

14. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Acid Fast Cell Wall – mainly Mycobacteria and Nocardia
Have a GP cell wall structure but also a waxy layer of glycolipids and fatty acids (mycolic acid)
Waxy layer makes them difficult to gram stain
Cannot be decolorized by acid-alcohol, hence the name “acid fast”

15. 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
Contain sterols in cell membrane

16. 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)

17. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Some bacteria produce slime layers
Made of polysaccharides
Inhibit phagocytosis
Aid in adherence to host tissue or implants

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

19. Examples of Flagella

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

21. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)

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

23. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Common Bacterial Stains
Gram Stain:
cell wall structure determines the staining characteristics
Procedure to be covered in lab
Acid-fast
stains bacteria with high lipid and wax content in their cell walls

24. 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

25. 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

26. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
India Ink – negative stain for capsules, surrounds certain yeasts

27. I NEED A BREAK!!!!!

28. 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

29. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Nutritional Requirements for Growth
Autotrophs (lithotrophs)
Able to grow simply, using only CO2, water and inorganic salts
Obtain energy via photosynthesis or oxidation of inorganic compounds
Occur in nature and do not normally cause disease

30. 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

31. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Types of Growth/Culture 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 which encourages small numbers of organisms to flourish
Broths- used to detect small numbers of aerobes, anaerobes and microaerophiles

32. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Nonselective medium- supports growth of most nonfastidious microbes.
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

33. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Selection of primary culture media will vary from lab to lab
Selection of primary media will depend on anatomical site

34. 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
Psychrophiles: cold temperatures
10-20o C
Mesophiles moderate temperatures
20-40 o C
Thermophiles: high temperatures
50-60 o C

35. 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
Microaerophilic- grow better in low oxygen environments ( about 20%)
Aerotolerant anaerobes- grows better in the absence of oxygen
Capnophilic – grow better with extra CO2 (5-10%)

36. 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 24 hours for M. tuberculosis

37. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Determination of Numbers
Direct counting under microscope
Estimates number of live and dead cells
Direct plate count
Determines the number of CFU (colony-forming units) in broth cultures and urine cultures
Density measurement
Useful to prepare inoculums for antimicrobial susceptibility testing

38. Is It Time For a Break?

39. 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

40. 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

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

42. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Metabolic Pathways
Embden-Meyerhoff-Parnas
Primary cycle for bacteria
Convert glucose to pyruvic acid, a key intermediate
Generates energy in the form of ATP
Pentose Phosphate pathway
Entner-Doudoroff pathway

43. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
From pyruvic acid:
Alcoholic fermentation (ethanol)
Homolactic fermentation (lactic acid)
Heterolactic fermentation (lactic acid, CO2, alcohols, formic and acetic acids
Propionic acid (propionic acid)
Mixed acid fermentation (lactic, acetic, succinic, and formic)
Butanediol fermentation(acetoin and 2,3 butanediol)
Butyric acid fermentation (butyric)

44. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Main oxidative pathway is the Krebs Cycle, resulting in acid and CO2
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

45. 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. This is how resistance is acquired.

46. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Plasmid replication
Three methods
Transformation
Transduction
Conjugation

47. Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
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

48. References
Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams and Wilkins.
Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.