Clemson Advanced Micro Lab Quiz 1

Use of high-pressure steam to sterilize liquids and certain labware items.

minimum conditions for autoclaving

Ovens set to a minimum of 160C for 2 hours

Physical process that literally removes microbes from suspension.

Common pore size for filtration

Flaming of loops and other materials to sterilize prior to use

target range for plate counts

So, if you are handed a culture and were told that it had 100,000,000 cfu/ml, what would your final dilution need to be to hit the range?

used for small to moderate volumes with the small volume method, syringe filter.

What materials would you use filtration for instead of autoclaving.

A liquid used to grow microbes

broth with a solidifying agent

solutions of material of unknown composition

solutions which the concentration of all materials within are known

media used to encourage the growth of organisms with specific capabilities

Good sanitation, proper PPE, proper disposal of biological wastes, reduction of aerosols and other means of transmission

aka laminar flow hoods, use HEPA-filtered air to provide a curtain of protection from stray contaminants, used for BSL2 or higher

Partial sunlight, some turbidity, decaying plant matter, healthy plant life

pH, temperature, salinity, turbidity, dissolved oxygen, nitrate, phosphate, total organic carbon, etc

require extra nutritional support in the form of a growth factor

Salt, sugar, blood, chocolate (lysed RBC)

pH, oxygen content, temperature, salt concentration, organic content

You could probably think of all microbiological media as selective. Why?

What does starting with a liquid culture system do?

Physical Growth Forms for media

Method to culture (partially) thousands of isolates

Make liquid extract from ecosystem Many small cultures (96 per plate) Automated

Allows for rapid assessment of biotech potential as well as the ability to acquire some physiological data

Little Ecosystems, Highly varied

Design suited to system under study

Common materials used and equipment adapted as necessary

grow faster

slower growth, grow to carrying capacity of land

Use of light to measure chemical concentration and/or makeup

Based upon the interaction of specific wavelengths with materials

Distance light travels through analyte

The amount of a specific wavelength of light is absorbed by a standard concentration of a chemical. 1 M in 1 cm pathlength

C = A/ε —> C is concentration, A is Absorbance (at X nm), and ε is the extinction coefficient at the defined wavelength.

Counting cells, finding concentration via a standard curve, determining the chemical makeup

not destructive, quick, easy to use, minimal data processing, inexpensive

Types of Centrifuge ranked smallest —> largest

Relationship between RPM & G-Force

at bottom, more dense

on top, less dense, liquid

Refrigeration is standard, allowing you to protect your samples from heat degradation.

The chamber is armored in case of rotor failure or operator error.

Excellent for separating modest amounts of cells from medi

Holds tubes of 50 ml max volume.

The pellet will be at the bottom, towards the outside of the rotor.

How should you load a centrifuge rotor?

allows you to use gradients more easily to separate materials.

To sort suspended particulates by size/density

How to report Centrifuge conditions

Units for speed, temperature of centrifugation

what does PCR stand for?

Key components of PCR

Equipment needed for PCR

General steps of PCR

DNA sample starts at room temp and heated

Primers bind to tell DNA pol where to start

DNA pol works - puts into place

Two major issue with PCR success

Goal for readings of DNA on spectrometer & ratio

Three methods of site-directed mutagenesis

Goal of protein tagging

site for antibodies to bind

affinity tag

chromatography tag

how much of X is in a DNA sample

determining the amount of nucleic acid present in a sample uses fluorophores

requirement of qPCR

technique used to monitor the presence of any RNA within the cell

Experimental design of Reverse Transcriptase PCR

Limit of Reverse Transcriptase PCR

different between RT-PCR and RT-qPCR

Methods of preservation

not well preserved, requires periodic passage

live cultures, held at 4 C, periodically passaged

Two least desirable methods of preservation and why

-80 to -120 C, addition of cryopreservative

Suitable Cryopreservative & goal

removal of water via sublimation

sublimation

requirement of lyophilization

limits of lyophilization

why are assays necessary?

growth, no growth, follow cellular responses, high throughput analysis streams

Things to consider in Biochemical assays

Things to consider in Cell based assays

Basic assay questions

changes in temp, pH buffer, cofactors, co enzymes

adapting and assay to a smaller volume

helper molecules, organic/inorganic, don’t bind

organic, bind to active site. substrate recruitment

Four types of assays

activity results in a change that can be measured immediately

activity that causes a change that can only be measured through a secondary reaction

a change in color is measured, requires a spectrophotometer

emitted light is measured, associated with ATP hydrolysis, requires fluorimeter

What do extinction coefficients tell you?

When is a standard curve still necessary?

equation for standard error

data near mean

data more spread out

how different a measure sample is from overall population

How do you think you can prove your technical measurement is valid? (hint: additional matching experiments)

Common equipment used. for assays