Bacterial Culturing Concepts and Techniques

Lab Safety and Technology and Lots of Microbiology Stuff

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.

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.

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.

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

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

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

Osmotic Pressure

Microbes obtain almost all their nutrients in solution from surrounding water

Tonicity isotonic hypertonic hypotonic

Cells

Oxygen

Bacteria can be classified base on their oxygen requirements Obligate Aerobes Obligate Anaerobes Facultative Anaerobes Microaerophilic

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)

2. Obligate Anaerobes 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

3. Facultative Anaerobes 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

4. Microaerophilic 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

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

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

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

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

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

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

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.

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

Phases of Growth

4 Phases

1. Lag Phase 2. Log Phase 3. Stationary

Phase 4. Death Phase

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

2. Log Phase

Rapid cell growth (exponential growth) population doubles every generation microbes are sensitive to adverse

conditions antibiotics anti-microbial agents

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?

3. Stationary PhaseEndospores 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?

4. Death Phase

Death rate > rate of reproduction Due to limiting factors in the environment

Bacterial Culture Techniques

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

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

AmplicillinVery stable antibioticThresholds for selection very broad

What does this mean? Inactivated by heat. Needs to be

added to media after it has cooled.

Bacterial Culture Techniques

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 wastesTreat with Bleach solution

Wipe down bench solution Wash hands before leaving lab