Lecture Topic 2 - Cultivation of Bacteria

Nutrients

Nutrients

all chemicals required by microorganisms are raw materials for their metabolism and reproduction

Two types of Mineral Nutrients

1. Micro-nutrients

2. Macro-nutrients

Examples of Micro-nutrients

1. cobalt

2. copper

3. manganese

4. molybdenum

5. nickel

6. selenium

7. tungsten

8. vanadium

9. zinc

Examples of Macro-nutrients

1. oxygen

2. hydrogen

3. carbon

4. nitrogen

5. phosphorus

6. sulfur

7. potassium

8. magnesium

9. sodium

10. calcium

11. iron

Growth factors

1. amino acids

2. purines and pyrimidines

3. vitamins (riboflavin, cobalamin, biotin, PABA, folic acid, niacin, thiamine, vitamin K, vitamin B6)

Classification of Microorganisms according to CARBON SOURCE

1. Autotroph: producers, photosynthetic, use CO and H2O, sunlight as energy, make their own food

2. Heterotroph: require preformed food, digestive and absorptive, most microbes

Classification of Microorganisms according to ENERGY SOURCE

1. Chemotrophs: Anaerobic Respiration - oxidative phosphorylation (oxidation of organic or inorganic compounds)

2. Phototrophs - Photophosphorylation (light as the principal source)

Classification of Microorganisms according to ELECTRON SOURCE

1. Lithotrophs: organisms that can use reduced inorganic compounds as electron donors

2. Organotrophs: organisms that can use organic compounds as electron donors

Physical Requirements for Microbial Growth - Temperature

1. Temperature - most important factor that determines the rate of microbial growth, multiplication, survival, and death of all microorganisms.

Effects of Temperature

1. very low temperature, metabolic rates are very slow

2. as the temperature rises, enzymatic reactions inside the cell proceed at faster rates and growth also becomes rapid.

3. subsequent increase in temperature may kill the microorganism

Classification of Microorganism according to TEMPERATURE Psychrophiles/Cryophiles

1. 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

1. grows best in moderate temperatures

2. temperature range: 20 - 45

3. most microorganisms are mesophiles

4. examples are: e. coli, s. pneumoniae, etc.

Classification of Microorganism according to TEMPERATURE Thermophiles

1. heat-loving microorganisms

2. grow best at temperature above 45

3. optimum growth is between 50 - 80

4. examples are: thermus aquaticus, geogemma barrosii, etc.

Classification of Microorganism according to TEMPERATURE Hyperthermophiles

1. thrives in extremely hot environment

2. temperature range: 80 - 113

3. the cell membrane contains high levels of saturated fatty acids to retain its shape at high temperatures.

Physical Requirements for Microbial Growth - pH

1. refers to the negative logarithm of Hydrogen ion concentration

2. microbial growth is strongly affected by the pH of the culture medium

Classification of Microorganisms according to pH

1. acidophiles - grow between pH 0 and 5.5

2. neutrophiles - grow between pH 5.5 - 8.0

3. alkalophiles - grow between pH 8 to 14

Physical Requirements for Microbial Growth - Oxygen and Carbon Dioxide

1. in order to cultivate microbial cells in the laboratory, the proper gas atmosphere must be present.

2. some gases are used in cellular metabolism, others may have to be excluded from a culture because they are toxic to the cells.

3. oxygen and carbon dioxide are the two principal gases that affect growth of microbial cells

Classification of Microorganisms according to OXYGEN

1. Aerobes - organisms that can survive and grow in an oxygenated environment.

2. Anaerobes - microorganisms which may be poisoned by oxygen, cannot grow in an air atmosphere

3. Facultative Anaerobes - anaerobic bacteria that do not require oxygen for growth although may use it for production if available.

4. 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

1. tolerant anaerobes - can tolerate low concentrations of oxygen

2. 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

1. pressure-dependent microbes

2. 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

1. to grow and isolate all bacteria present in an infection

2. 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

3. identification and characterization: to obtain sufficient growth of clinically relevant bacteria to allow identification and characterization

Basic Components of Culture Media

1. water (70-85%)

2. carbon

3. energy (0.5%)

4. nitrogen

5. mineral

6. growth factors

7. oxygen, moisture, pH, temperature

Types of Culture Media - According to Physical State or Consistency

1. liquid media - contain peptone and NaCl as major ingredients

2. solid media - can be prepared by adding agar into liquid media

3. semi - solid media - useful for the cultivation of micro

Types of Culture Media - According to Chemical Composition

1. simple media - contain only basic nutrients required for the growth of ordinary organisms and used as a general purpose media

2. complex media - contain some ingredients of unknown chemical composition

3. synthetic or chemically-defined media - prepared exclusively from pure chemical substances and their exact composition is known.

Basal Media

1. simplest and most common medium in diagnostic laboratories

2. support growth of non-fastidious bacteria

3. e.g. nutrient agar, nutrient broth, peptone water

Enriched Media

1. prepared to meet nutritional requirements of bacteria by the additional of substances like blood, chocolate, or serum to a basal medium

Enrichment Media

1. when a substances added to a liquid medium which inhibits the growth of unwanted bacteria and favors the growth of wanted bacteria.

2. usually liquid

Selective Media

1. 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

1. contain certain reagents or supplements which when incorporated into culture media may allow differentiation of various kinds of bacteria

Indicator Media

1. these media contain an indicator which changes color

Transport Media

1. a holding medium designed to preserve the viability of microorganisms in the specimen but not allow multiplication

Anaerobic Media

1. these media are used to grow anaerobic organisms and contain reducing substances

Types of Culture Methods

1. streak plate culture

2. lawn culture

3. stroke culture

4. stab culture

5. pour plate technique

6. 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

1. for cultivation of aerobes the incubation is done in an incubator under normal atmospheric condition

Anaerobic Culture

1. Anaerobic bacteria require incubation without oxygen and differ in their requirement and sensitivity to oxygen

Preserving Bacterial Cultures

1. Refrigeration

2. Deep freezing

3. Lyophilization (freeze drying)

4. Cold storage

5. Drying Methods