NRSG 205L Lab 8 - Bacterial Quantification (TWO)

In ideal conditions, how fast can a bacterial population expand?

At an exponential fate

With a generation time of ____ minutes, one E. Coli can turn into _______ in about _____ hours

20 minutes, 1 billion, 10

Exponential bacterial growth formula

2^n, with n the number of generations

Generation time

the time required for a population to double in size through binary fission

Generation time formula

G = t / (3.3 x log (Nt/N0)

Meaning of t, Nt, and N0 in generation time formula

• t = time

• Nt = number of colonies after a given time

• N0 = number of starting colonies

Colony forming unit (CFU)

measurement used to estimate the number of viable bacteria or fungal cells in a sample

A cell is only considered viable if what condition is met?

Cell is only considered viable if it is able to multiply via binary fission under the controlled conditions

How to calculate CFUs in two steps

1. Spread a specific volume of the microbial culture onto an agar plate

2. After incubating the plate, the number of colonies is proportional to the number of viable colonies in the tube

Formula to calculate CFUs

CFUs = # of colonies/volume spread

Units for CFUs

 CFU/mL

Because there are often too many colonies to count directly, what do we do?

You need to perform a serial dilution in order to decrease the number of cells that you use for spreading

How to calculate the CFU/mL of a diluted sample

You need to multiply the number of colonies with the dilution factor

Alternate technique for quantifying number of bacteria

Examination with a microscope

2 cons of quantifying bacteria with a microscope

1. Requires significant growth

2. Doesn’t exclude death or non-viable cells

Direct dilution formula

M1V1 = M2V2

Serial dilution formula

1/(final concentration/initial concentration)

What does serial dilution do?

Serial dilution splits up the dilution factor into multiple test tubes (such as 1000X into 10 X 10 X 10)

In serial dilution, for a dilution factor X, there is ___ part(s) solution, _____ part(s) diluent

1, X - 1

Antibiotic resistance

the general process in which bacteria - usually infectious - becomes insensitive to a specific drug or a specific class of drugs

When does antibiotic resistance typically appear?

When a microbe is exposed to sub-lethal concentrations of a specific antibiotic due to the selective pressure, the surviving microwaves might develop resistance mechanisms across several generations through DNA mutations

4 categories of resistance mechanisms

1. Modification of the primary target of the antibiotic

2. The inactivation of the antibiotic compound itself

3. Preventing the antibiotic from reaching the target, by reducing the permeability of the membrane or using efflux pumps to keep the drug outside the cell

4. Using alternative metabolic pathways to compensate for the action of the antibiotic and ensure the survival of the organism

Example of modification of the primary target of the antibiotic (resistance mechanism)

Transpeptidase for ampicillin resistance

Example of inactivation of the antibiotic compound itself (resistance mechanism)

Production of B-lactamase enzymes to degrade penicillin G inside the cell

Example of preventing the antibiotic from reaching the target (resistance mechanism)

Ciprofloxacin

Main concern of antibiotic resistance

transmission of these drug resistance across species

How is antibiotic resistance transmitted across species?

• A drug-resistant bacterial strain can transfer the resistance gene to other strains of the same species - or to individuals of a different species altogether - that have never been exposed to the drug in the first place

• This way, antibiotic resistance can spread widely and compromise whole antibiotic therapies

The accumulation of antibiotic resistance leads to the creation of what?

Superbugs - bacterial strains that are immune to all but the most drastic treatments

2 ways to overcome antibiotic resistance

1. Discover new antibiotics with novel modes of actions

2. Use different antibiotics in “cocktails” - presuming that a bacteria might survive facing one, but not 2 or 3 antibiotics simultaneously

In the lab, how are antibiotic resistance genes used?

In the lab, antibiotic resistance genes are an important tool for genetic engineering to select for transformed cells

Escherichia coli

rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms

Pathogenic qualities of some serotypes of E. Coli

Can cause serious food poisoning in their hosts, and are occasionally responsible for product recalls due to food contamination

2 benefits of resident E. Coli in the gut

1. Produces K2

2. Prevent colonization of the intestine with pathogeni bacteria

2 benefits of E. Coli in the lab

1. E. Coli can be grown and cultured easily and inexpensively in a lab setting, making it one of the best-studied model organisms

2. E. Coli is an important species in the fields of biotechnology and microbiology, where it has served as the host organism for the majority of work with recombinant DNA

Generation time of E. Coli

Generation time is only 20 minutes under favorable conditions