Prelab Questions BIO206L

For the final exam.

Bacteria cell wall function

protect cell from lysis

Gram - has, + doesn’t

outer membrane

crystal violet binds to

peptidoglycan

adding iodine solution produces

large crystals

Gram - bacteria turn pink/red when you stain with

safranin

gram staining, hot plate at:

2.5

slide removed from hot plate when:

water evaporates

post-evaporation heat-fixing time on hot plate

1 min

all dyes absorb for

1 min

decolorizer drips over sample for

up to 15 secs

gram staining procedure

crystal violet stain, iodine, alchohol, safranin

alcohol washing turns gram - bacteria _____

colorless

safranin turns gram - bacteria

pink

E. Coli is gram _ and morphology __

-, bacillus

ID the gram + Enterococcus

A

Morphology and Gram status

+, bacillus

MacConkey agar grows Gram

-

Gram-stained bacteria growing on MacConkey agar should be ____

pink

Why is gram staining differential

peptidoglucan cell wall

counterstaining in gram staining is for

enhancing contrast of stained bacteria

after crystal violet, if you forget iodine, what would happen?

gram + and - bacteria appear pink

align 10X objective with:

nosepiece

total magnification =

objective*10

better resolution is with 200 nm or 1000nm?

200 nm

micrometers per reticle unit and magnification are _______ related

inversely

biggest field of view with ___ objective

4X

biggest depth of field in focus with ___ objective

4X

can use coarse knob with ___ objective

4X

immersion oil for ___ objective

100X

100X objective has direct contact with immersion oil?

no

cleaning the objective lens/immersion oil:

use lens cleaner and lens paper

colors: gram - is ____, gram + is ______

red/pink, purple/violet

gram + bacteria are purple after staining because

bacteria retains the crystal violet-iodine complex

L-form/no cell wall bacterium is stained, what color?

pink

why do you gram stain bacteria

ID cell wall characteristics to help classify bacteria into broader taxonomic groups

ID the bacillus gram +, coccus gram +, and spirillum gram - bacteria

C, B, A

streptococcus, staphylococcus, or diplococcus?

streptococcus

week 9 lab purpose

verify soil isolates are bacteria via PCR and gel electrophoresis

PCR amplification enzyme

DNA Polymerase

what is not a step in PCR

sequencing

denaturation step temperature

95 C

what is NOT in the master mix:

primers

amplify means to _____

copy

The 27F and 1492R primers will anneal to _______, providing a starting point for Taq polymerase.

bacterial DNA

The DNA sequence amplified when the 27F and 1492R primers are used is approx ____ bp

1500

27S and 1492R primers are specifically for the _____ gene on _____ chromosomes

16S rRNA, bacterial

denaturation is

separating DNA strands

Taq polymerase synthesizes primers: T/F

F

T/F: Taq polymerase can only synthesize complementary DNA if it's given a primer.

T

What happens during the coolest stage of PCR?

Primers anneal to complementary seequences

Taq polymerase active temp

72 C

Expected contents of PCR tube when using bacterial chromosome:

millions of 16S rRNA gene sequence copies

Negative Control PCR tube post-reaction if there is NO contamination contains what?

no copies of the desired sequence

Expected Contents of Positive Control PCR Tube

amplified 16S rRNA gene

successful amplification shows as what in the gel?

1500 bp amplicons in a band

how do you confirm no contamination in gel electrophoresis

no band in negative control lane

positive control lane expected result:

1500bp band size

if bacteria can’t be lysed, then PCR reagents can’t access bacterial chromosome. will amplification happen? band?

neither will happen

agarose gel in electrophoresis does what:

separate DNA molecules by size

agarose gel electrophoresis procedure steps:

make solid gel, place in chamber, add buffer, load DNA ladder and all samples, apply current, measure the bands using the DNA ladder

determine reagents contamination using which lane?

negative control

is this the expected result

yes

which lane to check for expected result of gel electrophoresis

positive control lane

Isolate 1: bacteria or not? And how to confirm?

not bacteria, check if 1500bp band present in the lane

Isolate 2: bacteria or not?

bacteria

motility test - motile bacteria are ___ relative to the inoculation line

disperse from line/diffuse growth pattern

which is cytochrome c oxidase positive?

B

what to add to the oxidase reagent strip before bacteria

water

Which one is gram - (macconkey agar)

both

which ferments lactose?

B

MacConkey agar exigence

fecal matter contaminated drinking water

macconkey agar selects for enteric (gut) bacteria using

bile in the agar

E. Coli will grow/not grow and be what color?

grow, pink/red agar

E. raff will grow/not grow and be what color?

card response pending

P. Putida grow/not grow and if so what color?

grow, agar color doesn’t change

Gram + bacteria on macconkey agar: grow/not grow

not grow

Why does a clear zone surround beta-hemolytic bacteria?

bacteria break down red blood celss

hemolysis type?

alpha

hemolysis type?

beta

on blood agar, E. coli shows

clear zone

on blood agar, E. raff shows

clear zone

on blood agar, P. Putida shows

green

we used ____ blood agar plates

2

catalse + bacteria make ____ bubbles

O2

where do catalase test

fume hood

how much H2O2 in a catalase test sample

1-2 drops

catalase is important because

h2o2 damages cell components

E. raff on blood agar tests Catalase + falsely, because____

Catalase is in mammalian blood

oxidase test is for presence of:

aerobic respiration

catalase test is for

h2o2 breakdown

macconkey agar tests for

lactose fermentation/breakdown

motility test is for

flagella presence

hemolysis test is for

red blood cell breakdown

antibiotic resistance

bacteria grow despite drug that once prevented its growth

antimicrobial resistance mainly driven by

overuse

WHO fact shet reports ___% of UTIs from E. coli were less susceptible to ampicillin

20

The same gene that provides resistance to ampicillin spreads from a strain of E. coli to a different bacterial species (e.g., a Staphylococcus species.). How did this happen?

horizontal gene transfer

antimicrobial resistance occurs naturally too: T/F

true

this mechanism doesn’t drive antibiotic resistance:

Antibiotic inactivation by enzymes

1. Decreased cellular uptake

2. Antibiotic target mimicry

3. Bacterial colony signaling

bacterial colony signaling

bacterium has efflux pump exporting multiple different antimicrobial drugs, therfore giving it multiple resistances, AKA

cross-resistance