Chapter 6. Growth and Culturing of Bacteria

 

·         Definition of growth

·         Unicellular organisms increase in size to approximately two times the original size.  At that time the mother cell divides into two daughter cells by binary fission.  The daughter cells grow - become mother cells and divide.  With each division, the population doubles.  Microbial growth is defined in terms of increase in cell number rather than size.

·         Cell division occurs by binary fission or in some cases (yeast, for example) by budding.  Figure 6.2

·         The doubling in cell number requires replication of the chromosome as well as other components of the cell.

 

·         Phases of growth

·         Microbial growth curve - lag phase, log phase or exponential growth phase, stationary phase, decline or death phase. Figure 6.3

·         Associate unique events with each phase.

·         Generation time - the time required for the population to double.

·         Determination of generation time.

·         Gt= log10 Nf – log 10 Ni / 0.301

·         Number of generations = T/Gt

·          

·         Measuring bacterial growthTechnique of serial dilution - Figure 6.6

·         One bacterium results in one bacterial colony.  Figure 6.7

·         Countable number of colonies (30 to 300 per plate) - Figure 6.8.

·         Determination of cell number viable vs physical count Figure 6.9

·         Estimating cell mass by spectrophotometry - take advantage of turbidity - Figure 6.11

·         Compare methods as to viability of the bacteria

 

·         Factors affecting the growth of bacteria

·         Physical Factos:

·         pH - negative logarithm of the hydrogen ion concentration of an aqueous solution - Figure 2.7

·         Temperature - Figure 6.13

·         psychrophiles obligate (Bacillus globisporus), facultative (Xanthomonas pharmicola), <20oC, but usually greater than 15

·         mesophiles - 25 to 40oC - thermoduric organisms can survive elevated temperatures, but grow best at moderate temperatures.

·         thermophiles – 40 to 650 C.

·         Extreme thermophiles - > 650 C.

·          oxygen Figure 6.15

·         obligate aerobes

·         obligate anaerobes

·         facultative anaerobes

·         microaerophiles

·         capnophiles - microaerophiles that are CO2 loving

·         aerotolerant anaerobes

·         moisture

·         all microorganisms require an aqueous environment at most or all stages of the life cycle

·         osmotic pressure

·         most microorganisms are relatively tolerant of the external environment relative to the osmolarity or tonicity.

·         halophiles grow only in high salt concentrations - Figure 6.16.

·         Halophiles survive because they can regulate osmotic pressure by packing inside the cells “compatible solutes”

·         Nutritional Factors

·         the growth of microorganisms is affected greatly by the need for nutrients

·         some microbes are fastidious - need specific nutrient requirements - others are quite nonfastidious

·         nutrient requirements are very useful for the differential identification of microorganisms.

·         Macronutrients: Carbon, Nitrogen, Sulfur, Phosphorus

·         Trace Elements – mineral ions

·         Vitamins

 

Sporulation

 

·         Sporulation is the formation of endospores  and occurs in the genera Bacillus and Clostridia.  A few other genera also have this capability.

·         When nutrients become limiting, primarily carbon and nitrogen sources, endospores begin to form in the mother cells.

·         Endospores are not metabolically active and are highly resistant to drought, temperature extremes, radiation and some toxic chemicals.

·         Endospores cannot divide and the parent cell can produce only a single endospore – thus sporulation is not a mechanism of reproduction – only survival.

·         Figure 6.16: 

·         As sporulation begins, the DNA replicates and forms a long axial nucleoid.

·         The two chromosomes move to different locations within the cell.

·         The DNA where the endospore will form directs the formation of the endospore.

·         Most of the cell’s RNA and some of the cytoplasmic proteins condense around the DNA and form the core of the spore. 

·         The core also contains dipicolinic acid and calcium ions – probably contribute to the spore’s stability by stabilizing protein structure.

·         An endospore septum grows around the spore – a double membrane.

·         Peptidoglycan  is synthesized and fills the space between the membranes.

·         A spore coat of keratinized protein is laid down around the double membrane or cortex.

·         The spore is released to survive in the environment.

·         Under favorable conditions, the spore can germinate.

·         Germination usually requires some type of trauma such as heat or low pH to damage the coat and initiate activation. 

·         The spore then germinates into a vegatative cell which can grow and divide.

·         Examples of spore-formers - Figure 6.17.

·         Clostridium botulinum  - causes botulism

·         Clostridium tetanii - causes tetanus

·         Bacillus anthracis - causes anthrax

·         Endospores as old as 300 years have been successfully germinated.

·         Sporulation is a very effective mechanism of bacterial survival by a relatively few species.

 

Culturing of Bacteria

·         Pure cultures - concept is: one bacterium gives rise to one colony

·         Streak plate method - Figure 6.18

·         Pour plate method - Figure 6.6

·         Culture media

·         solid medium vs liquid medium

·         agar is added to a liquid medium to solidify it - Frau Angelina Hesse the American wife of one of Pasteur’s colleagues suggested adding agar to liquid media to solidify it.  This enabled Koch to grow bacteria in pure cultures.

·         minimal medium - chemically defined medium -Table 6.2

·         complex medium - chemically non-defined medium Tables 6.3, 6.4

·         nutrient agar

·         blood agar

·         chocalate agar

·         MacConkey’s agar

·         etc.

·         Selective media encourages the growth of certain organisms and discourage others

·         Differential media enables different species to be distinguished from each other

·         Enrichment media that encourage the growth of a specific organism

·         Preservation of cultures

·         cultures can be preserved by several procedures including lyophilization, storage frozen in a cryoprotective agent such as glycerol, in stab cultures or slants

·         Reference cultures are used as standards to identify cultures and to preserve specific characteristics.

 

 

In summary the growth and division of microorganisms requires a carefully choreographed arrangement of a large number of processes.  Little things can be exceedingly complex.