Clemson Advanced Micro Lab Quiz 1

Autoclaving

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

121 C, 15 psi, 15 min/liter

minimum conditions for autoclaving

Dry Heat

Ovens set to a minimum of 160C for 2 hours

Filtration

Physical process that literally removes microbes from suspension.

0.45 um, 0.22 um

Common pore size for filtration

Incineration

Flaming of loops and other materials to sterilize prior to use

30-300

target range for plate counts

10^-6

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?

Filter Sterilization

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

Heat labile

What materials would you use filtration for instead of autoclaving.

Broth

A liquid used to grow microbes

Solid

broth with a solidifying agent

Complex Media

solutions of material of unknown composition

Defined Media

solutions which the concentration of all materials within are known

Enriched Media

media used to encourage the growth of organisms with specific capabilities

BSL1

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

Biosafety Cabinets

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

Visual Observations

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

Measured Observations

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

Fastidious

require extra nutritional support in the form of a growth factor

Growth Factor

Salt, sugar, blood, chocolate (lysed RBC)

Enrichment Parameters

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

No Universal Medium

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

Increase cell abundance

What does starting with a liquid culture system do?

plate, deep tube, broth, slant

Physical Growth Forms for media

High Throughput culturing

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

Microcosms

Little Ecosystems, Highly varied

Design suited to system under study

Common materials used and equipment adapted as necessary

r Strategists

grow faster

k strategists

slower growth, grow to carrying capacity of land

Spectroscopy

Use of light to measure chemical concentration and/or makeup

Based upon the interaction of specific wavelengths with materials

Pathlength

Distance light travels through analyte

Extinction Coefficient

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

Beers Lambert Law

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

Uses of Spectroscopy

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

Spectroscopy Strengths

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

micro < preparative < ultra

Types of Centrifuge ranked smallest —> largest

Proportional

Relationship between RPM & G-Force

Precipitate (pellet)

at bottom, more dense

Supernatant (supernate)

on top, less dense, liquid

Preparative Centrifuge

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

Fixed Angle Rotors

Holds tubes of 50 ml max volume.

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

same orientation, balanced weights

How should you load a centrifuge rotor?

Swinging Bucket Rotor

allows you to use gradients more easily to separate materials.

Differential Centrifuge

To sort suspended particulates by size/density

speed, temperature, time, buffer/gradient composition

How to report Centrifuge conditions

xg, C

Units for speed, temperature of centrifugation

polymerase chain reaction

what does PCR stand for?

DNA primers, DNA polymerase, DNTPs, Buffer, DNA sample

Key components of PCR

thermal cycler, PCR tubes

Equipment needed for PCR

Denaturation, Annealing. Extension/Elongation, Final Elongation, Final hold

General steps of PCR

Denaturation

DNA sample starts at room temp and heated

Annealing

Primers bind to tell DNA pol where to start

Extension/Elongation

DNA pol works - puts into place

DNA purity, Phenolic Contamination

Two major issue with PCR success

260/280 (DNA/Protein), 1-8-2.2 ratio

Goal for readings of DNA on spectrometer & ratio

1 in 1 out, remove base, add x3

Three methods of site-directed mutagenesis

manipulate DNA to add to it with recombinant protein to purify

Goal of protein tagging

epitope tag

site for antibodies to bind

protein will have affinity to stick to things

affinity tag

sticks to specific parts of the chromatography column

chromatography tag

qPCR

how much of X is in a DNA sample

RT-qPCR

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

control DNA of the same size

requirement of qPCR

Reverse Transcriptase PCR

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

ssRNA —RT—> dsDNA —> PCR

Experimental design of Reverse Transcriptase PCR

you need to know what you are looking for

Limit of Reverse Transcriptase PCR

Methodology

different between RT-PCR and RT-qPCR

live cultures, refrigeration, freezing, lyophilization

Methods of preservation

Live cultures

not well preserved, requires periodic passage

Refrigeration

live cultures, held at 4 C, periodically passaged

Live cultures, Refrigeration, due to genetic change build up

Two least desirable methods of preservation and why

Freezing

-80 to -120 C, addition of cryopreservative

10-15% glycerol, prevent ice crystals

Suitable Cryopreservative & goal

lysophilization

removal of water via sublimation

solid —> gas

sublimation

high vacuum, high SA to volume ratio , frozen material

requirement of lyophilization

long process

limits of lyophilization

gather/confirm quantitative data about a process & rates

why are assays necessary?

Biological Assays

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

pH, temp, Ion concentration, reagent solubility/stability/aggregation, order of reagent addition, instrumentation

Things to consider in Biochemical assays

biochemical considerations + cell culture plastics, media, conditions, serum, cell cycle, passage number

Things to consider in Cell based assays

What are you detecting, how would you measure it,does an assay already exist

Basic assay questions

Adapting an assay

changes in temp, pH buffer, cofactors, co enzymes

Miniturization

adapting and assay to a smaller volume

Cofactors

helper molecules, organic/inorganic, don’t bind

Coenzymes

organic, bind to active site. substrate recruitment

direct, indirect, colorimetric, fluorescent

Four types of assays

Direct Assay

activity results in a change that can be measured immediately

Indirect Assay

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

Colorimetric Assay

a change in color is measured, requires a spectrophotometer

Fluorescent Assay

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

Directly qualify a compounds without a standard curve

What do extinction coefficients tell you?

When relating concentration of indicator to another compound

When is a standard curve still necessary?

SE = SD/SQRT(n)

equation for standard error

high standard deviation

data near mean

Low standard deviation

data more spread out

Standard error

how different a measure sample is from overall population

Biological repetition

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

UV & Visible Spectrophotometers, Fluorimeters, HPLC, Gas chromatography, Gel Electrophoresis, Microscopy

Common equipment used. for assays