bio lab quiz 1

bacteria

variable in shape, color texture, opacity, colony, size

yeast

convex, generally dry and opaque

mold

often fuzzy made of hyphae

bacteria divereged

before split between archea and eukaryotes

over a period of time there is a lot of variablility that has developed

methods to diffeeneciate bacterial species

dna sequencing of a portion of 16s RNA

morphological and biochemical tests

16s rRNA is good for sequencing becuase

it evoleves slowly and good for primer design. small portions tolerate mutations and evolve quickly

selective medium and differential medium

allows some bacteria to survive, then tells us about the additional properties of the bacteria that lived

mannitol is fermentated

is yellow/orange

biochemical assays

oxidase and catalase

oxidase

tests for the presence of cytochrome C oxidates involved in respiration

bacteria that test positive are AEROBIC

tests negative means it cant use oxygen for electron acceptor

catalase

all organism possess defense mechanism to repare or escape the oxidative damage caused by hydrogen pyroxide

visible bubbles test positive

we use two positive controls - catalase and oxidase

becuase ensure our reagants are working and since its difficult to judge the result

pcr

generate billions of copies of small segment of an organism genome for further analysis

denature—> Anneal—> extend

gram positive

thick peptidoglycan layer

gram negative

thin peptidoglycan layer

forward and reverse primers

these are short segments of synthetic ingle stranded dna that we constructed with known sequences

pcr master mix

taq dna polymerase

dNTPs , MgCl2

buffers to maintain pH

anneal

temp is lowered to a point where each primer binds to its target on single stranded dna

the forward primer binds to one strand and the reverse primer binds to other

16S RNA

universal primers. they flank a region that is variable in nucleotide sequence in different bacterial species

growth on mannitol salt agar (MSA)

high concentration of NaCl that most bacteria cannot survive on

salt tolerant species

fermenting changes the surrounding to a red/organce/yelow

catalase test

enzyme catalse present in some bacteria will break down its substrate, H2o2 into o2 and h2o

positive test forms bubbles

oxidase test

bacteria hat test positive must be aerobic

species tghat test negative cant use o2 as electron acceptor and use something different to transfer electrons to oxygen

artificial electron acceptor

colorless to darj blue or purple when recieves and electron

KOH string test

gram positive bacteria have thick outer wall

stains purple when positive

negative stains pink

gram positive WIILL NOT FORM STRINGS when touching KOH

3% KOH

Peptidoglycan Layer

Provides structural support and protect on.

Thickness influences susceptibility to antbiotics

Phenylethyl Alcohol Agar (PEA):

inhibits gram - negative bacteria by interfering with DNA synthesis due to the thick peptidoglycan layer

Eosin Methylene (EMB) agar

contains methylene blue which inhibits gram positive bacteria,

Calculation example

If concentrate on is 50 ng/µl, maximum volume for 15 ng target is 0.3 µl

Nano Spectrophotometer

measures DNA concentration with only 2 microliter sample

procides absorbance readings to calculate conentration

requires careful calibration and backround correction

sequencing pcr

prepare master mix , addd 3-10 ng of purified pcr product

mix throughrouly and keep on ice

master mix components

dNTPs, ddNTPs,Taq polymerase, buffers , salts

What is the role of BB buffer

Buffer BB contains salts that facilitate binding of the PCR-amplified DNA to the filter in the bottom of the spin column

role of WB buffer

Buffer WB is a buffer containing ethanol and is used to wash away impurities

role of EB buffer

The EB buffer will release the pure DNA from the membrane and the DNA will flow through the column