BSCI223 Exam 2

General Bacterial Enumeration

General:

• Relies on sampling

  • cells are evenly distributed

  • accuracy depends on samples being neither too dense nor too diluted

• Expressed as:

  • cells/ml or CFU/ml (colony forming units)

  • cells/g or CFU/g

  • cell/m² or CFU/m²

  • ABS

• Cells/ml and CFU/ml are equal in single, free living cells that can form colonies (ex: E.coli)

  • not equal for all organisms, because some organisms form pairs, chains, etc

Ways to Enumerate Bacteria

1. Viable Plate Count

   • only count living cells

   • plates, spreaders, incubators, etc

   • takes time (colonies)

   • start with original suspension, perform serial dilutions (usually 1:10 dilutions → dilutions have neg exponents, CFU/ml will almost always have positive exponents) in order to dilute to a countable amount, each tube is 10 fold less the concentration, and then plate tube that isn’t too dense and isn’t too dilute (tube can produce sampling error from too many colonies or too little colonies)

   • normally put around 0.1 ml onto plates (can give sampling error if too little = dries out or too much = takes too long to dry)

2. Direct Counts

   • immediate results after counting

   • requires counting chamber and microscope

   • cells need to be immobile

   • counts living and dead cells

   • count within glass grid (might need staining, but can’t tell if cells are dead or alive)

3. Spectrophotometry

   • immediate results (very fast results)

   • requires spectrophotometer

   • counts living and dead cells

   • gives density, not CFU/ml

   • 0 absorbance = 100% transmittance of light (cells will absorb some of the light when present)

COUNTABLE PLATES: 30-300 colonies

1. Original Sample Density:

   • Divide all dilutions (including plating dilutions)

   • 0.1 ml is 1/10 of 1 ml

   • 0.1 ml plating dilution is a 1:10 dilution

   • # colonies from 1 plate/total dilution

Microbial Growth

• Refers to cellular replication

1. Binary Fission

   • One cell becomes two cells

   • Most bacteria replicate this way

2. Budding

   • Yeasts

   • parental cell differentiates into parental cell and progeny cell (progeny cell usually smaller than the parental cell) and then detaches from the parental cell (still smaller), and then it grows in size to match that of parental cell size

3. Filamentous Growth

   • Fungi and some bacteria growth

   • more cells made

   • can have branching, maintained connection

Replication

1. Single cell with cytoplasmic membrane and chromosome bundled up, cell wall

2. Chromosomes replicated throughout entire cycle, but cell ends up with two copies of same chromosome, which are then separated

3. mechanisms to allow for symmetric division (asymmetric division can be fatal to one or both cells)

4. Chromosomes move to polar ends

5. Septum formed between two cells, connecting cytoplasmic membranes and cell walls

6. Can happen multiple times (exponential growth)

7. Generation time = time required for a population to double in number (doubling time), consistent between numbers

Phases of Growth in Batch Culture

1. Lag Phase = synthesis, adaptation to environment (kicking up metabolism)

   • number of cells not changing

2. Exponential or log Phase = rapid increase in cell numbers

   • actively dividing cells

   • depends on media, temperature, organism, etc

3. Stationary Phase = lack of available nutrients prohibits growth (media is no longer rich enough to increase growth)

   • no net increase in numbers

   • cell division rate = cell death rate

   • cells are still dividing

   • cells are using each other for growth

4. Death phase = decrease in viable cell numbers

   • stretch out stationary phase long enough and can see this

   • not present for all organisms

   • death > doubling

   • senescence → slowed down physiology

Growth Curve + Primary Metabolites

1. primary metabolite synthesis

   • essential for life (amino acids, DNA bases, vitamins, etc)

   • synthesized before division

   • DNA replication

Growth Curve + Secondary Metabolites

• provide cells with selective advantages under some conditions

• nonessential for life, but provide advantage (antibiotics)

• cost cell energy investment to make

• useful for eliminating competition for resources

• synthesis occurs when nutrition is depleted in media and when cell number increasing

Growth Curve + Sporulation

• spores form as conditions worsen

• spore = dormant cell type that allows cells to survive adverse conditions until conditions improve

Growth Measurement

1. Log Scales

   • useful when measuring rapid changes

   • each tick is 10x different

     • 10² = 100

     • 10³ = 1000

   • Never gets to zero

2. Linear Scales

   • normal numbering, many ticks, but leads to many numbers that around the same amount being bunched up on top of one another → doesn’t visibly show minor differences between numbers

Growth Condition Comparison

• log shows differences in data, while linear tends to clump data together and is harder to read at low time

Calculating Doubling Time

• take two points on log graph (line of best fit) where CFU/ml doubles

• subtract between the two times to calculate doubling time

Phases of Growth for Batch Culture

• Determine OD required to grow a particular CFU/ml:

  • draw line from CFU/ml to time, then draw line from same time to where that time occurs on the OD line, then draw line to y axis of OD plot to determine OD required

Heat as Control Method

• incineration

• effects of high temperatures (above max temp)

  • denatures proteins

  • interferes with integrity of membranes and cell wall

  • disrupts structures and functions of nucleic acids

• Thermal death point = lowest temperature that kills all cells in broth in 10 mins

• Thermal death time (TDT) = time to sterilize volume of liquid at set temperature

  • has to be above maximum temperature, but hotter culture = shorter death time

• Decimal reduction time = time to achieve 10x reduction in number over a given time

  • one log loss of cells (lose 90% of cells)

  • lower temperature, but still above max temperature = extend decimal reduction time

  • higher temperature above max temperature = lowering decimal reduction time (die faster)

Cow

• cattle breakdown feedstocks (cellulose) mechanically into smaller pieces to allow it to be digested later

• cattle rumen → main part of cellulose digestion

  • rumen has huge population of microbes

    • cellulolytic bacteria, methanogens (generate CH4), etc

  • cellulolytic → process of breaking down cellulose into more simple sugar

  • very little oxygen = fermentation

  • products converted to volatile fatty acids = bacterial waste products absorbed by rumen wall, which feeds cows

• methane produced, which can trap heat in the environment significantly more efficient than CO2

• methanotroph = consumes methane

  • methane comes into membrane, converted into methanol via methane monooxygenase, oxygen, and 2 protons → creates alcohol group

  • by products can be converted into cell carbon or forms formate (aldehyde to formate produces NADH and formate to CO2 produces another NADH)

  • functional ETC and can make ATP

Central Dogma

• phenotype = all measurable characteristics a cell

  • shape, color, structures, motile, etc

  • proteins do most of phenotype work

• genotype = all genetic elements of a cell

  • gives rise to phenotypes

  • chromosomes

  • DNA is source of genotype

• genes (DNA) → RNA → proteins

  • DNA molecule always larger than RNA molecule, RNA molecule always larger than protein, etc

  • all read left to right

• information is organized on chromosomes

• genes go in both directions

  • DNA double stranded, so genes can go from left or right

  • very rarely → genes overlap

  • can have long stretches of genes on one strand, but none on the other strand

• gene name → lacZ, pmoA

  • DNA that makes up gene

• protein name → LacZ, PmoA

• protein name based on function → B-galactosidase, Particulate Methane Monooxygenase

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