1. Microbiology- a clinical approach by Anthony Strelkauskas et al. 2010 Chapter 10: Bacterial growth

2.  Increase in numbers is one of the requirements for infection. ◦ This increase is dependent upon bacterial growth.  Understanding the requirements for growth will help in understanding the infectious process and collection techniques for clinical specimens.  Specific techniques used by clinical microbiologists have a key role in identifying and diagnosing bacterial diseases.

4.  Each division of bacteria is called a generation.  The time between divisions is called generation time.  Some pathogens have excessively long generation times while others have very short generation times.  The shorter the generation time, the faster the number of bacteria increases within the host.

5. For comparison: in vitro human epithelial cells have a generation time of ~ 36 hours; during this time 1 E. coli would have undergone 72 cell divisions leading to 2 72 = ~ 4 x 10 21 bacteria.

6.  Infectious organisms have specific growth requirements. ◦ Temperature ◦ pH ◦ Osmotic pressure ◦ Atmosphere (oxygen!) ◦ Nutrients  These specific requirements allow for a maximum increase in numbers of infectious organisms.  Increased numbers of pathogens help to defeat the host defense. physical chemical

7.  Bacteria are found in all ranges of temperatures.  Bacteria can be separated according to temperature ranges in which they grow best. ◦ Psychrophiles – grow at cold temperatures ◦ Mesophiles – grow at moderate temperatures ◦ Thermophiles – grow at high temperature  Extreme temperature requirements are typically found among archaea. ~ -5 -15C ~ 10 – 45 C ~ 40 – 80 C

8.  Temperature affects growth. ◦ Increased temperature breaks chemical bonds.  This causes changes in the three dimensional structure.  These changes can inhibit or destroy the ability for the molecules to function properly. ◦ Decreased temperature slows down chemical reactions, movement of proteins through membrane.

9.  Most human pathogens are mesophiles, whereby variable temperature requirements are seen in certain diseases.  Treponema pallidum (the causative agent of syphilis) likes lower temperatures. ◦ Lesions are first seen on exterior parts of the body including lips, tongue, and genitalia.  Mycobacterium leprae (the causative agent of leprosy) also likes lower temperatures. ◦ Lesions are first seen on the extremities of the body including the face, ears, hands, feet, and fingers.

10. Primary syphilis Leprosy

11.  Bacteria grow in a wide range of pH values.  Most bacteria prefer the neutral pH of 7.0.  Some bacteria are acidophiles that grow at extremely low pH values. They require a low pH for their growth. ◦ Helicobacter pylori (causes stomach and duodenal ulcers) grows at a low pH value but is not an acidophile. It secretes an enzyme (urease) that increases the pH.  Some bacteria are alkaliphile. They require a high pH for their growth.

12.  pH can negatively affect protein structure. ◦ An excess of hydrogen ions causes bonds to break and make amino acids positively charged (NH 4 + –R) ◦ This changes three-dimensional structure and binding pockets and destroys protein function.  Destruction of protein function can be a lethal event.  Phagocytes induce a drop in pH in lysosomes upon fusion with the phagosome.

13.  Osmotic pressure is the pressure exerted on bacteria by their environment.  One of the major agents exerting such pressure is solute concentration.  Halophilic organisms can be divided into: ◦ Obligate – requiring a high salt concentration ◦ Extreme – requiring very high levels of salt (5- 6 M NaCl) ◦ Facultative or tolerant– can grow either with or without elevated salt levels

14.  High salt concentrations can be used to preserve food (cure meats). ◦ Cause a hypertonic environment and plasmolysis. ◦ This is an imperfect way to preserve food because some bacteria are halophilic and require and thrive in high salt concentrations. Plasmolysis

15.  Adapted to human body. ◦ Optimal osmotic pressure ◦ Optimal temperature range ◦ Optimal pH range  The human body serves as an excellent incubator for pathogens.

16. A. extreme thermophile B. mesophile C. thermophile D. psychrophile E. psychotroph

17.  The chemical requirements are almost as variable as bacterial species themselves.  Many of the chemical requirements for bacteria are the same as for human cells.  Several core chemicals are required for bacterial growth.

18.  All biological molecules contain carbon.  Bacteria are classified based on ways that they acquire carbon. ◦ Chemoheterotrophs – obtain carbon by breaking down other carbon molecules ◦ Chemoautotrophs – obtain carbon from CO 2.

19.  Nitrogen is involved in protein synthesis. ◦ It is an integral part of amino acid structure.  It is part of the structure of nucleic acids.  It can be obtained in a variety of ways: ◦ Decomposition of existing proteins ◦ From ammonium ions found in organic materials ◦ Nitrogen fixation

20.  Bacteria must have sulfur to make amino acids and some vitamins.  Sulfur is obtained from decomposition.  Sulfur can be procured in the sulfate ions (SO 4 2- ) and from H 2 S.

21.  Phosphorus is essential for the synthesis of nucleic acids, AMP, ADP, and ATP.  It is a major component for the development of the plasma membrane.  Bacteria obtain phosphorus by cleaving ATP or ADP or from phosphate ions.

22.  Bacteria use growth factors such as vitamin B 1, B 2, and B 6.  Bacteria cannot synthesize these growth factors so they must be obtained from the environment.  Bacteria also require potassium, magnesium, and calcium as enzyme co-factors.  Some bacteria also require trace elements such as iron, copper, molybdenum, and zinc.

24.  Oxygen spontaneously generates toxic radicals such as superoxide free radicals.  Bacteria differ in the expression of the matching detoxifying enzymes. ◦ Superoxide dismutase, catalase  If bacteria express these detoxifying enzymes they can withstand oxygen.  If they lack these enzymes they will die.

25. O 2 - + O 2 - + 2H + H 2 O 2 + O 2 2H 2 O 2 2H 2 O + O 2 SOD Catalase

26.  There are three major categories of bacteria based on oxygen use: ◦ 1. Obligate aerobes – require oxygen for growth ◦ 2. Anerobes - obligate anaerobes cannot survive in the presence of oxygen; facultative anaerobes can grow with or without oxygen. ◦ 3. Aerotolerants grow in the presence of oxygen but do not use it; Microaerophiles: require reduced concentrations of oxygen. Gas gangrene left foot

27.  Fluid sodium thioglycolate medium forms an oxygen gradient during growth. ◦ (1) Anaerobic organisms grow at the bottom. ◦ (2) Aerobic organisms grow at the top. ◦ (3) Facultative anaerobes grow throughout the medium. oxygen

28.  The second method for growing anaerobic organisms is in a GasPak TM jar ◦ This incubation container provides an oxygen-free environment through chemical reactions. ◦ Only obligate and facultative anaerobes can grow via this method.

29.  hyper-thermophile; grows between 80°C and 130°C  acidophile; grows between pH 0 and pH 5  mesophile; grows between 25°C and 45°C  microaerophile; grows in 20% oxygen  alkalophile; grows between pH 8.5 and pH 11.5

31.  Growth media must provide all of the essential growth factors.  Some bacteria are considered to be fastidious. ◦ They require a large number of these growth factors rather than just one or two growth factors. ◦ They grow very slowly and can be missed diagnostically.  There are two types of growth media: ◦ Chemically defined media ◦ Complex media

32.  Growth media can be used to identify pathogens in several ways.  A selective medium is one that contains ingredients that prohibit the growth of some organisms while fostering the growth of others.  A differential medium is one that contains ingredients that can differentiate between organisms.  Many selective media are also differential media.

33.  Bacteria divide primarily by binary fission ◦ The parent cell divides into two daughter cells.  Each division is considered a generation.  The time between divisions is the generation time.  Generation times vary between bacterial species and are heavily influenced by: ◦ Temperature ◦ Environmental pH ◦ Osmotic pressure ◦ Availability of nutrients ◦ Oxygen level

36.  Lag phase – bacteria are adjusting to their environment ◦ This varies depending on the organisms and the environment.  Log phase – the number of bacteria doubles exponentially ◦ There is a constant minimum generation time. ◦ This phase lasts only as long as a suitable level of nutrients is available. ◦ Bacteria are the most metabolically active and most susceptible to antibiotics.  Stationary phase – the number of cells dividing is equal to the number dying ◦ It is caused by a decreasing availability of nutrients. ◦ Many virulence genes are up-regulated during this phase  Death phase (logarithmic decline phase) – a continuous decline in the number of dividing cells ◦ It is caused by the exhaustion of the nutrient supply and by a build-up of metabolic waste.

37.  There are two ways to measure bacterial growth: ◦ Direct ◦ Indirect  Direct methods of measuring bacterial growth include: ◦ Direct cell counts ◦ Viable cell counts  Plate counts  Membrane filtration  Indirect methods of measuring bacterial growth include: ◦ Turbidity – most often used ◦ Measuring cell products

39.  Many bacteria are fastidious in a laboratory setting.  Some bacteria cannot be grown in a laboratory setting.  Some bacteria have stringent nutritional requirements.  All of these things can affect diagnosis and treatment of infection.

40.  Bacterial growth requirements can lead to missed identification of pathogens and wrong diagnoses ◦ This can be caused by improper handling of clinical specimens. ◦ It can also be cause by improper culturing.  Specific standard procedures for collecting specimens are intended to limit these problems.

42.  Bacterial growth is an important part of the infection process.  Growth of bacteria requires proper physical and chemical environments.  Different bacteria have different requirements for oxygen. Obligate anaerobic organisms cannot grow in the presence of oxygen, aerobic organisms cannot grow without it, and facultative anaerobes can grow with or without oxygen.  Identification of organisms is important for the study of bacteria and the treatment of disease.  Media, either complex or chemically defined, can be used to grow bacteria in a laboratory setting.

43.  Selective medium prohibits the growth of some organisms while fostering the growth of others.  Differential medium contains ingredients that can differentiate between organisms.  Most bacteria grow by the process of binary fission.  Different bacteria have different generation times.  The bacterial growth curve has four phases: lag, log, stationary, and death.  Different methods are used to take samples from different parts of the body.