Growth vs. Tolerance
– Growth - referring to the number of cells, not the size.
– Tolerance - survive under conditions in which they cannot grow.
– The suffix “-phile” describe conditions permitting growth, whereas the term “tolerant” describes conditions in which the organisms survive, but don’t necessarily grow.
– E.g. a “thermophilic bacterium”, “thermotolerant bacterium”
The requirements for growth
• Physical requirements
- light, temperature, pH, water activity and osmotic pressure.
• Chemical requirements
- electron donor (C, N, S, P, K)
- electron acceptor (O2 - , NO3- , SO4 2-, CO3 - , Fe3+) - micronutrients (vitamins, amino acids, trace minerals)
Trace elements - e.g. Fe, Mg, Mn, Cu, Zn …
- Microorganism dependant
- Important for enzyme function
- part of enzyme and cofactors
- catalysis of reactions
- maintenance of protein structure
- Organic growth factors Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines
Special growth factors
- Specifically needed for growth of certain m/os
- e.g. Legionella pneumophila (Legionaires’ disease)
~ absolute requirement for a.a. L-cystein and iron
~ in nature
~ provided by algae and amoebas
Aseptic technique
Definition Aseptic technique refers to carrying out a procedure under controlled conditions in a manner that will minimize the chance of contamination. Contaminants may be introduced from the environment, equipment and supplies, or personnel.
Aseptic Technique
• Sterile Hood - All manipulations must be carried out in a sterile cabinet – Turn the UV light off (Should ordinarily be on) – Open the cabinet – Wipe down with disinfectant (70% ethanol or 40% isopropyl alcohol)
• Bring materials into the hood
• Light up the flame or gas
• Begin your work
• Flame all caps and lids
• Tightly close all boples and caps
• Remove materials from the hood
• Turn off gas
• Wash the hood surface
• Turn the UV light on to disinfect
Culturing Microorganisms
• There are two basic culture techniques used in microbiology:
1. Liquid culture: bacteria, algae, and some fungi can be reared in culture tubes (test tubes) in a liquid medium.
~ Liquid medium is best when you want to rapidly increase the concentration of the organism or when you want to grow motile cells.
2. Culture Plates: Liquid medium is solidified using agar (agarose) and poured as a thin layer in the bopom of a culture dish (also some=mes called petri plate)
~ Culture plates are used when you want to test (1) antibiotic sensitivity, (2) estimate culture concentrations from environmental samples, or (3) isolate individual colonies from environmental samples
Culturing bacteria
• Culturing bacteria in the laboratory present two problems:
–To obtain pure culture
–To use suitable medium
Pure culture
• Is a population of identical cells originating from a single cell.
• Pure cultures are obtained by working in aseptic environments.
Anaerobic Environments
• Reducing Compounds
– Thioglycholate
– Cystein
– Anything with – SH
– Must Use Indicator
• Gas Pack (anaerobic generating kit)
Anaerobic Incubation
• Anaerobic Jar
– Impermeable to Oxygen
• Catalyst
– Platinum or Palladium
– In Lid or on Gas Pack
• Gas Pack
– Uses Oxygen and Replaces with Carbon Dioxide
New technique to provide anaerobic environment
• Oxyrase
– Reduces oxygen to water
– Respiratory enzyme derived from the plasma membranes of certain bacteria
– Added into growth medium
• Avoid the need for more cumbersome apparatus
Culture media
CULTURE MEDIUM
- A nutrient material prepared for the growth of microorganisms in a laboratory.
INOCULUM
- Microbes that are introduced into a culture medium to initiate growth.
CULTURE
- The microbes that grow and multiply in or on a culture medium.
STERILE
- No living microbes
Types of Culture Media
• Chemically defined vs. complex media
– Chemically defined media
• The exact chemical composition is known • e.g. minimal media used in bacterial genetics experiments
– Complex media
• Exact chemical composition is not known
• Oten consist of plant or animal extracts, such as soybean meal, milk protein, etc.
• Include most routine laboratory media, e.g., tryptic soy broth.
• Liquid (broth) vs. semisolid media
– Liquid medium
• Components are dissolved in water and sterilized
– Semisolid medium
• A medium to which has been added a gelling agent
• Agar (most commonly used)
• Gelatin
• Silica gel (used when a non-organic gelling agent is required)
• Selective media
– Contain agents that inhibit the growth of certain bacteria while permiting the growth of others
– used to isolate specific organisms
• Differential media
– Contain indicators that react differently with different organisms (for example, producing colonies with different colors)
– Used in identifying specific organisms
• Enrichment media
– The enrichment media will increase the small numbers of desired bacteria to detectable level.
– One type of bacteria present in small numbers while the other type present in much larger numbers. – The enrichment medium is usually liquid and provides nutrients and environmental conditions that favor the growth of a particular microbe but not others.
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