BMS 503 - Bacterial Growth Control (Exam II)

physical techniques (optical density, direct microscopic counts, colony forming unit assays) and molecular-based techniques (quantitative PCR, Flow Cytometry)

How can you measure population growth?

turbidity or cloudiness of a sample that can be measured with a spectrophotometer

What is optical density?

the approximate number of bacteria in a sample

Optical density of a culture can be used to calculate what?

it gives an estimation of cell concentration and cannot tell the difference between viable and non-viable cells and can vary significantly based on cell morphology

What are the major drawbacks of optical density?

Using a counting chamber, bacteria are placed on a slide containing grids of a known area. The constant volume of liquid held by the slide allows of the concentration of cells per unit volume

How is direct microscopic count performed?

not

Direct microscopic count is ____ commonly used in the lab

A culture of bacteria is serially diluted in sterile liquid media, each dilution is plated on sterile agar plate, the number of colonies on the plate indicates the number of viable cells in the original culture, and the cell numver is represented as a colony forming unit per mL (CFU/mL)

How does CFU assay work?

efficient amplification of specific DNA sequences in the laboratory

What does PCR allow for?

template DNA, dNTPs, DNA polymerase, and Primers

What are the reactions included in PCR?

backbone, DNA sequencing, Molecular cloning, Clinical Diagnostics

PCR technology is one of the _____ of modern research, used commonly for what procedures?

exponential as a function of the number of cycles in the reaction

PCR products grow how?

proportional

In PCR, the amount of a given DNA sequence in a culture is ____ to the number of bacteria in a culture

a PCR-based technique that couples amplification of a target DNA sequence with quantification of the concentration of that DNA species in the reaction.

What is quantitative PCR?

first, the amplified DNA is fluorescently labeled (usually with cyanine-based fluorescent dyes), and second, the amount of fluorescence released during amplification is directly proportional to the amount of amplified DNA

How does qPCR work?

how the storage of milk affected the bacterial load, allowing scientists to monitor the increase of the DNA concentration of bacteria

In the Yersinia enterocolitica qPCR experiment, what was examined?

temperature, oxygen concentration, pH, hydrostatic pressure, osmotic pressure

What are the physical factors in addition to nutrient levels that affect bacterial growth?

broad (ex. E. coli grows optimally at pH 7, but can grow well in pH range 5.5-8)

Often organisms with have a _____ range of environmental conditions in which they can occur

above 80 C (in sea floor hot water vents)

Hyperthermophiles grow in what temperature?

between 50-80 C (best at 60 C, in compost heaps and hot springs)

Thermophiles grow in what temperature?

between 15-45 C

Mesophiles grow in what temperature?

below 15 C (make up largest portion of prokaryotes on Earth)

Psychrophiles grow in what temperature?

above 9

Alkaliphiles grow in what pH?

between 5 and 8

Neutralophiles grow in what pH?

below 3

Acidophiles grow in what pH?

high salt concentrations

Halophiles grow in what?

only oxygen

Aerobes grow in what?

with or without oxygen

Facultative aerobes grow how?

in small amounts of oxygen

Microaerophiles grow how?

without oxygen

Anaerobes grow how?

at high pressure greater than 380 ATM

Barophiles grow how?

in pressures between 10-495 ATM

Barotolerants grow how?

matches

A bacterial cell's temperature ___ that of its immediate environment

optimum, minimum and maximum

Each organism has an ____ temperature, as well as a ___ and ___ temperature tha define its growth limits

higher rates of growth

Microbes that grow at higher temperatures can typically achieve what?

the tendency of a biological system to maintain internal stability, owing to the coordinated response of its parts to any situation or stimulus that would tend to disturb its normal condition or funtion

What is homeostasis?

heat-shock response

Rapid temperature changes experienced during growth activates bactes of stress response genes that result in what?

protein shape, change the membrane lipid composition

The protein products include chaperones that maintain what and enzymes that do what?

insensitive to oxygen

What are aerotolerant anaerobes?

inhibited or killed by oxygen

What are obligate anaerobes?

require an atmosphere low in oxygen and rich in carbon dioxide

What are capnophilic bacteria?

organisms's oxygen sensitivity

Thioglycollate broth can be used to test what?

in top of broth where high concentrations of oxygen have diluted into the medium

Obligate aerobes will grow in a thioglycollate broth how?

growth occurs throughout tube, but best where oxygen is most present (top of medium)

Facultative anaerobes will grow in a thioglycollate broth how?

growth occurs where there is no oxygen (bottom of medium)

Obligate anaerobes will grow in a thioglycollate broth how?

growth occurs evenly, oxygen has no effect

Aerotolerant anaerobes will grow in a thioglycollate broth how?

growth occurs only where a low concentration of oxygen has diluted into medium (middle of medium)

Microaerophiles will grow in a thioglycollate broth how?

a closed jar and a lit candle that reduces oxygen concentration

What is a candle jar?

acandlee is lit and placed in a jar sealed with cultures, the candle flame reduces oxygen until flame dies, creates a reduced oxygen environment with increased CO2

What does a candle jar do?

a jar that creates an oxygen-free environment

What is an anaerobic jar?

pumping in a mixture of H2 and CO2 gas

How is the O2 in an anaerobic jar replaced?

using a gazpak and palladium catalyst

How is the O2 removed from an anaerobic jar?

gaspak generates H2 which in the presence of palladium catalyst will combine with oxygen to produce water

How does the gazpak and palladium catalyst work?

optima, minima, maxima

All enzyme activities exhibit ___, ____ and ____ with regard to pH

regulate their internal pH to maintain homeostasis

When the environment is in a similar pH range to the internal pH of the cell, bacteria can what?

disrupt cell pH homeostasis, killing cells

Weak acids pss through membranes and do what?

Microbes prevent the unwanted influx of protons by exchanging extracellular K+ for intracellular H+ when the internal pH is too low, or cells use Na+/H+ antiporter to bring protons into cell when pH is too high

How does pH homeostasis work?

equalize

A solution of solute and solvent separated by a permeable membrane will always try to____

the diffusion of fluid through a partially permeable membrane from an area of low solute concentration to an area of high solute concentration

What is osmosis?

osmotic pressure

Cells are bags of solute and environments are filled with solute, causing a pressure to be put on the cell membrane called what?

osmolarity, membranes

All cells, including bacteria, have to maintain their internal solute concentrations called ____ to maintain integrity of their _____

destruction of all microbes, spores, and viruses (become contaminated when exposed to air and surroundings)

What is sterilization?

reducing the numbers of pathogens of discouraging their growth

What is sanitation?

physical procedures or chemical agents used to destroy microbes or render them inert

What is disinfection?

high, mid, and low

What are the three levels of disinfection?

used for items involved in invasive procedures (anything that sterilizes)

What is high level disinfection?

used for items or surfaces that are not likely contaminated with spores or highly resilient microorganisms, used for non invasive instruments (breathing apparatus)

What is mid level disinfection?

used to treat noncritical items (i.e. blood pressure cuffs)

What is low level disinfection?

heat, radiation, filtration

What are the three most common physical methods/procedures used for disinfection?

proteins and nucleic acids are destroyed adn water is removed

At temperatures above the growth range, what happens?

thermal death time

Each microbial species has a _____ for a given temperature

thermal death point

What is the minimm temperature at which all bacteria in a culture will die within 10 minutes?

decimal reduction time (DRT)

Heat resistant bacteria are described by the amount of time it takes to reduce a culture by 90% at temperature X known as what?

dry heat

___ requires long periods of exposure to high temperatures

the heat changes microbial proteins and removes water (desiccation). An example is incineration (extreme)

What does dry heat do?

dry heat ovens

_____ are used for sterilization utilizing high temperature over long periods of time

moist heat

_____ is faster and effective at a lower temperature than dry heat

denatures and coagulates cell proteins after 10 min

How does moist heat work?

applying steam heat of 121 C at 15 PSI for 15 min: items placed in, air in chamber is evacuated by vacuum pump, steam is forced into chamber at high temperature

How do autoclaves work?

sterilize medical waste and infectious materials, culture media, surgical tools, tattoo needles, lab glassware and sterility is determined by indicator tape

How are autoclaves used?

plastics

What CANNOT be autoclaved?

traps microorganisms by passing fluid/air through a filter that traps organisms above a certain size in pores (HEPA filter, useful for liquids that could be destroyed by heat)

What does filtration do?

UV and ionizing

What are the two types of radiation?

crosslinks nucleic acids rendering organism unable to replicate genetic material

What does UV irradiation do?

create strand breaks in nucleic acids rendering organism unable to replicate genetic material

What does ionizing irradiation do?

spores with intrinsic resistance to low level radiation, endospores, viruses, or protein toxins

Radtiation is active against most microorganisms except what?

with gamma rays from decay of cobalt-60 or cesium 137

Irradiation is used in food preservation how?

drying (removes water), salting/curing (causes dehydration and death), low temperatures (lowers microbial metabolic growth rates)

Besides irradiation, what other methods are important to food preservation?

antiseptics and disinfectants

What are the two categories of chemical disinfection?

chemical agents that are used to kill microbes on living tissues

What are antiseptics?

chemical agents that are used to kill microbes on a non living surface

What are disinfectants?

kill or slow growth of microbes, be non toxic to animals, be soluble in water or alcohol, be easy to apply, have a relatively long shelf life, be used in diluted form, act in short time, be nonstaining and non corrosive, be odorless and easy to obtain, be relatively inexpensive

Ideally, a disinfectant or antiseptic should be able to:

indicates disinfecting ability compared to that of phenol (varies by agent and organism)

What is a phenol coeffecient?

ethylene oxide gas, formaldehyde gas, oxidizing agents, chlorine

What are the high chemical disinfectants?

alcohol and iodine

What are the medium chemical disinfectnats?

quarternary ammonium compounds, soap and water

What are the low chemical disinfectants?

a high chemical disinfectants that alkylates proteins, disrupting function, that requires training and a specialized chamber (explosive)

What is ethylene oxide gas?

a high chemical disinfectant that alkylates proteins, disrupting function, that requires training and a specialized chamber (carcinogenic)

What is formaldehyde gas?

a high chemical disinfectant that destroys proteins and DNA (reactive oxygen species) that includes hydrogen peroxide, peracetic acid, and ozone

What is an oxidizing agent?

a high chemical disinfectant that destroys proteins, membranes, and nucleic acids as a highly reactive oxidizer that includes hypochlorous acid and bleach

What is chlorine?

a medium chemical disinfectant that disrupts membrane bi-layers and includes isopropanol and ethanol at or above 70% (below is ineffective)

What is alcohol?