microbio lab practical

first step of gram stain

primary stain, reagent is crystal violet, gram pos and gram neg is purple

second step of gram stain

mordant, reagent is iodine solution, gram pos and gram neg purple

third step of gram stain

decolorization, reagent is alcohol, gram pos purple, gram neg clear cause don’t have enough peptidoglycan to retain purple

fourth step of gram stain

counterstain, reagent is safranin, gram pos purple, gram neg pink

purpose of gram stain

To differentiate bacteria based on cell wall composition

results- purple

is gram positive and has thick peptidoglycan

results- pink

is gram negative and has thin peptidoglycan and outer membrane

bacillus subtilis 

bacteria, gram positive, motile

citrobacter freundii

bacteria, gram negative, motile

Ecoli K12

bacteria, gram neg, motile

enterobacter aerogenes 

bacteria, gram neg, motile

enterococcus durans

bacteria, gram positive, nonmotile

micrococcus luteus

bacteria, gram pos, nonmotile, pigmented

micrococcus roseus

bacteria, gram positive, nonmotile

pseudomonas fluorescens

bacteria, gram negative, motile, pigmented

S cerevisiae 

fungi, nonmotile, yeast

Staphylococcus epidermidis

bacteria, gram positive, nonmotile

differentiation

used to distinguish one organism from another

selection

enhances the growth of one organism while suppressing another organism 

hypothesis

what you think will happen, includes if then statement, preview of discussion section

predictions

describe appearance of results that would support the hypothesis, preview of results section

results

only what you can see or observe 

conclusions

if it was successful or not successful, interpretation of results, sources of error, what needs to be done next time

puc119 cut with ecori 

one band, 3.2kb 

puc119 with hindiii

one band, 3.2 kb

puc119 cut with ecori and hindii

one band, 3.2 kb

prux492 cut with ecori

one band, 3.2 kb plus x

prux492 cut with hindiii

one band, 3.2 kb plus x

prux492 cut with ecori and hindiii 

two band, 3.22 kb and x 

tryptone

provides amino acids or nitrogen source

yeast extract

provides vitamins

lactose

provides carbon source and sugar

mannitol

type of sugar and gives carbon source

dipotassium phosphate 

provides buffer and keeps stable ph in media 

sodium chloride

provides ideal osmotic pressure in media

dextrose

help microbes make atp

peptone

provides amino acids, peptides, and nitrogen

P10 micropipette 

from .5 to 10 ul 

P100 micropipette

from 10 to 100 ul

P1000 micropipette

from 100 to 1000 ul

p10 is 10 ul, p100 is 100ul, p 1000 is 1000ul

p10 is .5 ul, p100 is 10 ul, p1000 is 100ul

p10 is 3.2 ml, p100 is 54.5 ml, p1000 is 945ml

DH5a strain

is sensitive to ampicillin and can’t ferment lactose, agar is maroon and no colonies

DH5a transformed with pUC119

ferments lactose and ampicillin resistant, agar is maroon with maroon colonies

DH5a transformed with pRU4×92

can’t ferment lactose and amp resistant, agar is amber with clear colonies

transformation 

the genotypic and phenotypic alteration of a cell due to uptake of foreign dna 

plasmid isolation

removal of plasmid dna from previously transformed cells

ingredient important for selection

ampicillin cause it allows transformed cells to grow

ingredient important for differentiation

Ph indicator cause maroon colonies ferment lactose and clear colonies don’t

macconkey agar

selects for gram neg and differentiates between lactose fermenting and non lactose fermenting

What does it mean when the Macconkey agar turns from maroon to amber? 

the cell can not ferment lactose and is ampicillin resistant

What does it mean when you observe maroon colonies on maroon agar? 

the cell can ferment lactose, acid produced, ph dec