Bacterial Culturing Concepts and Techniques

1. Bacterial Culturing Concepts and Techniques Lab Safety and Technology and Lots of Microbiology Stuff

2. Microbiology Safety Guidelines • All materials (bookbags, clothing) other than laboratory manual and notebook should be off of the lab bench. NO EATING! • Sponge off the lab bench with the disinfectant solution (commercial product or 10% bleach solution) BEFORE and AFTER lab activities. • Wash hands BEFORE and AFTER lab activities with hand soap. • DO NOT PIPETTE WITH MOUTH. Use pipette bulb that is provided.

3. Microbiology Safety Guidelines • Discard used glassware (test tubes, petri dishes) in designated container- DO NOT PUT IN WASTE CAN. • Discard plastics, cotton swabs and other disposables in autoclave bags. • Because organisms used in class are potentially pathogenic, aseptic technique is important. Keep hands and other objects (pens, pencils) OUT OF YOUR MOUTH. DO NOT LICK LABELS- use drops of tap water.

4. Microbiology Safety Guidelines • Report any accidents that involve cuts, burns, or spilled cultures. If you should spill a culture, • Place the culture tube in its holder. • Place paper towels over the spill and liberally apply disinfectant solution to the towels. • Wearing gloves, dispose of the paper towels after several minutes into the designated plastic bag container. • Wash your hands with hand soap.

5. Microbial Growth - refers to the # of cells, not the size of the cells • Because individual cells grow larger only to divide into new individuals, microbial growth is defined not in terms of cell size but as the increase in the number of cells, which occurs by cell division. • This emphasis has practical application since it is typically far easier to measure increases in cell number than it is to measure increases in cell size • Furthermore, unless cell division is synchronized, cells will typically vary in size across an even homogeneous population, thus making measurement of cell size almost irrelevant as a means of measuring growth

6. Physical Requirements • Temperature • psychrophiles (cold loving microbes ) • range 0 C - 20 C • mesophiles (moderate temp. loving microbes) • range 20 C - 40 C • thermophiles (heat loving microbes) • range 40 C - 100 C

7. pH • Most bacteria grow between pH 6.5 - pH 7.5 • Very few can grow at below pH 4.0 • many foods, such as sauerkraut, pickles, and cheeses are preserved from spoilage by acids produced during fermentation

8. Osmotic Pressure • Microbes obtain almost all their nutrients in solution from surrounding water • Tonicity • isotonic • hypertonic • hypotonic

9. Cells

10. Oxygen • Bacteria can be classified base on their oxygen requirements • Obligate Aerobes • Obligate Anaerobes • Facultative Anaerobes • Microaerophilic

11. 1. Obligate Aerobes • An organism that has an oxygen based metabolism. Aerobes, in a process known as cellular respiration, use oxygen to oxidize substrates (for example sugars and fats) in order to obtain energy. • Organisms that are unable to generate ATP via fermentation are termed obligate aerobes • This term is somewhat misleading because some of these organisms can still grow in the absence of molecular oxygen by employing alternative final electron acceptors to their electron transport systems • The bottom line, then, for an obligate aerobe is a dependence on an electron transport system for their generation of ATP as well as a tolerance for atmospheric oxygen (which otherwise can serve as a poison)

12. Organisms that are unable to detoxify atmospheric oxygen are termed obligate anaerobes because they cannot grow (nor, often, even survive) in the presence of oxygen Obviously, obligate anaerobes must possess means for ATP generation that do not require molecular oxygen, e.g., fermentation pathways 2. Obligate Anaerobes

13. A facultative anaerobic organism is an organism that makes ATP by aerobic respiration if oxygen is present but is also capable of switching to fermentation. These organisms tend to exist in environments in which oxygen concentrations are uncertain, and serve as the oxygen scavengers in environments displaying relatively low oxygen concentrations For example, the lumen of the large intestine is mostly anaerobic because (i) the body does not actively oxygenate the lumen of the large intestine and (ii) oxygen scavengers such as Escherichia coli remove what oxygen manages to leak into this environment Facultative anaerobes tend to grow better/faster when O2 is present 3. Facultative Anaerobes

14. Organisms that require oxygen to survive, but requires environments containing lower levels of oxygen than are present in the atmosphere (~20% concentration). Typically much less than atmospheric concentrations, but more than those concentrations tolerable by obligate anaerobes Growth is inhibited by normal oxygen concentrations (ideal at approximately 200 μM). Nanoaerobes are organisms that cannot grow in the presence of μM concentrations of oxygen, but can grow with and benefit from nM concentrations of oxygen 4. Microaerophilic

15. Culture Media All microorganisms require the following nutrients to grow, repair themselves, and to replicate: Carbon, Nitrogen, Sulfur, Phosphorus, various trace elements • In addition, some microorganisms require various vitamins as well as additional organic factors (e.g., specific amino acids) • Although we are concerned with ways microorganisms satisfy their own nutritional needs, we can note that in satisfying such needs, they also help recycle elements in the environment. • That is, microorganisms are typically able to obtain nutrients from sources that macroorganisms are not • Fastidious - Microorganisms whose nutritional needs are unusually complex are termed fastidious

16. Culture Media • 1. Chemically Defined • the exact chemical composition is known • used to grow fastidious organisms • 2. Complex Media • exact chemical composition is not known • most bacteria and fungi are grown with this

17. Culture Media • Broth versus solid media • Broth media is liquid while solid media typically has agar added as a solidifying agent • Semi-solid media also exists that contains insufficient quantities of agar to fully solidify the media • Synthetic medium • A synthetic medium is prepared in the laboratory from reasonably well-defined ingredients • By contrast, a non-synthetic (authentic) medium could be something like soil or sewage or ocean mud, i.e., something obtained directly from the environment • Defined synthetic medium • A defined synthetic medium is produced only from well-defined, relatively pure ingredients

18. Culture Media • Complex media (chemically undefined media) • A second approach to producing a synthetic medium is to employ ingredients that are not well-defined nor pure • Such ingredients additionally may vary from batch to batch • For example, complex media may contain extracts from animals (e.g., beef, hearts, milk, etc.), plants (e.g., soy beans), or microorganisms (e.g., yeast) • Complex media may additionally include very complex ingredients such as blood or serum

19. Selective Media • Inhibits the growth of some bacteria while selecting for the growth of others • Example: • Brilliant Green Agar • dyes inhibit the growth of Gram (+) bacteria • selects for Gram (-) bacteria • Most G.I. Tract infections are caused by Gram (-) bacteria

20. Differential Media • Differentiates between different organisms growing on the same plate • Example: • Blood Agar Plates (TSA with 5% sheep blood) • used to differentiate different types of Streptococci

21. Bacterial Growth - increase in the # of cells • Binary Fission • Generation Time (Doubling Time) • time required for a cell to divide • most about 1 Hr. To 3 Hrs. • E. coli - 20 minutes • Mycobacterium tuberculosis - 24 Hrs.

22. Limiting factors in the environment • Lack of food, water or nutrients • space • accumulation of metabolic wastes • lack of oxygen • changes in pH • temperature

23. Phases of Growth • 4 Phases • 1. Lag Phase • 2. Log Phase • 3. Stationary Phase • 4. Death Phase

24. 1. Lag Phase • Bacteria are first introduced into an environment or media • Bacteria are “checking out” their surroundings • cells are very active metabolically • # of cells changes very little • 1 hour to several days

25. 2. Log Phase • Rapid cell growth (exponential growth) • population doubles every generation • microbes are sensitive to adverse conditions • antibiotics • anti-microbial agents

26. 3. Stationary Phase • Death rate = rate of reproduction • cells begin to encounter environmental stress • lack of nutrients • lack of water • not enough space • metabolic wastes • oxygen • pH Endospores would form now. What is an endospore?

27. 3. Stationary Phase Endospores would form now. What is an endospore? • a dormant, tough, non-reproductive structure • The primary function of most endospores is to ensure the survival of a bacterium through periods of environmental stress. • Why would they form now?

28. 4. Death Phase • Death rate > rate of reproduction • Due to limiting factors in the environment

29. Bacterial Cell Lines Most protocols have been tested and optimized with E. coli strains MM294 & MM294/pAMP very commonly used. pAMP means it contains a plasmid that gives the cell resistance to amplicillin which is an antibiotic Why would you want an amplicillin resistant strain of bacteria? Bacterial Culture Techniques

30. Nutrient Agar LB (Luria-Bertani) agar very commonly used Recipe for LB Add the following to 800ml H2O 10g Bacto-tryptone 5g yeast extract 10g NaCl Adjust pH to 7.5 with NaOH Add 15g agar Melt agar into solution in the microwave Adjust volume to 1L with dH2O Sterilize by autoclaving Bacterial Culture Techniques

31. Amplicillin Very stable antibiotic Thresholds for selection very broad What does this mean? Inactivated by heat. Needs to be added to media after it has cooled. Bacterial Culture Techniques

32. Handling and disposal of live bacterial strains Re-flame all tools when finished. Keep face away from pipette tip or loop Incubate plates only long enough for experiment than dispose properly Disinfect materials when experiments are over Autoclave wastes Treat with Bleach solution Wipe down bench solution Wash hands before leaving lab Bacterial Culture Techniques