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Nutrients
all chemicals required by microorganisms are raw materials for their metabolism and reproduction
Two types of Mineral Nutrients
Micro-nutrients
Macro-nutrients
Examples of Micro-nutrients
cobalt
copper
manganese
molybdenum
nickel
selenium
tungsten
vanadium
zinc
Examples of Macro-nutrients
oxygen
hydrogen
carbon
nitrogen
phosphorus
sulfur
potassium
magnesium
sodium
calcium
iron
Growth factors
amino acids
purines and pyrimidines
vitamins (riboflavin, cobalamin, biotin, PABA, folic acid, niacin, thiamine, vitamin K, vitamin B6)
Classification of Microorganisms according to CARBON SOURCE
Autotroph: producers, photosynthetic, use CO and H2O, sunlight as energy, make their own food
Heterotroph: require preformed food, digestive and absorptive, most microbes
Classification of Microorganisms according to ENERGY SOURCE
Chemotrophs: Anaerobic Respiration - oxidative phosphorylation (oxidation of organic or inorganic compounds)
Phototrophs - Photophosphorylation (light as the principal source)
Classification of Microorganisms according to ELECTRON SOURCE
Lithotrophs: organisms that can use reduced inorganic compounds as electron donors
Organotrophs: organisms that can use organic compounds as electron donors
Physical Requirements for Microbial Growth - Temperature
Temperature - most important factor that determines the rate of microbial growth, multiplication, survival, and death of all microorganisms.
Effects of Temperature
very low temperature, metabolic rates are very slow
as the temperature rises, enzymatic reactions inside the cell proceed at faster rates and growth also becomes rapid.
subsequent increase in temperature may kill the microorganism
Classification of Microorganism according to TEMPERATURE Psychrophiles/Cryophiles
able to grow at 0 degree celsius or lower but they grow best at higher temperatures (range: -20 degree celsius to 10 degree celsius)
Classification of Microorganism according to TEMPERATURE Mesophiles
grows best in moderate temperatures
temperature range: 20 - 45
most microorganisms are mesophiles
examples are: e. coli, s. pneumoniae, etc.
Classification of Microorganism according to TEMPERATURE Thermophiles
heat-loving microorganisms
grow best at temperature above 45
optimum growth is between 50 - 80
examples are: thermus aquaticus, geogemma barrosii, etc.
Classification of Microorganism according to TEMPERATURE Hyperthermophiles
thrives in extremely hot environment
temperature range: 80 - 113
the cell membrane contains high levels of saturated fatty acids to retain its shape at high temperatures.
Physical Requirements for Microbial Growth - pH
refers to the negative logarithm of Hydrogen ion concentration
microbial growth is strongly affected by the pH of the culture medium
Classification of Microorganisms according to pH
acidophiles - grow between pH 0 and 5.5
neutrophiles - grow between pH 5.5 - 8.0
alkalophiles - grow between pH 8 to 14
Physical Requirements for Microbial Growth - Oxygen and Carbon Dioxide
in order to cultivate microbial cells in the laboratory, the proper gas atmosphere must be present.
some gases are used in cellular metabolism, others may have to be excluded from a culture because they are toxic to the cells.
oxygen and carbon dioxide are the two principal gases that affect growth of microbial cells
Classification of Microorganisms according to OXYGEN
Aerobes - organisms that can survive and grow in an oxygenated environment.
Anaerobes - microorganisms which may be poisoned by oxygen, cannot grow in an air atmosphere
Facultative Anaerobes - anaerobic bacteria that do not require oxygen for growth although may use it for production if available.
Microaerophiles - cannot withstand the level of oxygen present in the air (21%) and usually grow best at oxygen levels between 1% and 15%.
Types of Anaerobes
tolerant anaerobes - can tolerate low concentrations of oxygen
strict anaerobes - killed by brief exposure of oxygen
Physical Requirements for Microbial Growth - Hydrostatic Pressure
pressure exerted on the cells by the movement of water resting on top of them
Classidication of Microorganims according to HYDROSTATIC PRESSURE Barophiles
pressure-dependent microbes
organisms that need a high-pressure environment in order to grow
Bacterial Cultivation
a method of multiplying microorganisms by letting them grow and reproduce in artificial culture media under controlled laboratory conditions or set-up.
IN - VITRO
inoculating the bacteria in a pre-determined culture medium and growing in a laboratory controlled environment
Main Purposes of Bacterial Cultivation
to grow and isolate all bacteria present in an infection
infection and contaminants or colonizers: to determine which of the bacteria that grow are most likely causing infection and which are likely contaminants or colonizers
identification and characterization: to obtain sufficient growth of clinically relevant bacteria to allow identification and characterization
Basic Components of Culture Media
water (70-85%)
carbon
energy (0.5%)
nitrogen
mineral
growth factors
oxygen, moisture, pH, temperature
Types of Culture Media - According to Physical State or Consistency
liquid media - contain peptone and NaCl as major ingredients
solid media - can be prepared by adding agar into liquid media
semi - solid media - useful for the cultivation of micro
Types of Culture Media - According to Chemical Composition
simple media - contain only basic nutrients required for the growth of ordinary organisms and used as a general purpose media
complex media - contain some ingredients of unknown chemical composition
synthetic or chemically-defined media - prepared exclusively from pure chemical substances and their exact composition is known.
Basal Media
simplest and most common medium in diagnostic laboratories
support growth of non-fastidious bacteria
e.g. nutrient agar, nutrient broth, peptone water
Enriched Media
prepared to meet nutritional requirements of bacteria by the additional of substances like blood, chocolate, or serum to a basal medium
Enrichment Media
when a substances added to a liquid medium which inhibits the growth of unwanted bacteria and favors the growth of wanted bacteria.
usually liquid
Selective Media
when a substance is added to a solid medium which inhibits the growth of unwanted bacteria but favors the growth of wanted bacteria
Differential Media
contain certain reagents or supplements which when incorporated into culture media may allow differentiation of various kinds of bacteria
Indicator Media
these media contain an indicator which changes color
Transport Media
a holding medium designed to preserve the viability of microorganisms in the specimen but not allow multiplication
Anaerobic Media
these media are used to grow anaerobic organisms and contain reducing substances
Types of Culture Methods
streak plate culture
lawn culture
stroke culture
stab culture
pour plate technique
liquid culture
Quadrant Streak Plate Method
this method is routinely employed for the isolation of bacteria in pure culture from clinical specimens
Lawn Culture Method
lawn cultures are prepared by flooding the surface of the plate with a liquid culture or suspension of the bacterium, pipetting off the excess inoculum and incubating the plate
Stroke Culture Method
stroke culture is made in tubes containing agar slope or slant. slopes are seeded by lightly smearing the surface of agar with loop in a zigzag pattern taking care not to cut the agar
Stab Culture Method
a suitable medium such as nutrient gelatin or glucose agar is punctured with a long, straight, charged wire into the center of the medium and withdrawing it in the same line to avoid splitting the medium
Liquid Culture Method
liquid cultures in tubes, bottles or flasks may be inoculated by touching with a charged loop or by adding the inoculum with pipette or syringes
Aerobic Culture
for cultivation of aerobes the incubation is done in an incubator under normal atmospheric condition
Anaerobic Culture
Anaerobic bacteria require incubation without oxygen and differ in their requirement and sensitivity to oxygen
Preserving Bacterial Cultures
Refrigeration
Deep freezing
Lyophilization (freeze drying)
Cold storage
Drying Methods