lab practical 2

what type of bacteria traps the crystal violet-iodine complex, which results in the purple color?

gram positive

in what type of bacteria does the thinner peptidoglycan and outer membrane allow the complex to be washed out, resulting in the pink color?

gram negative

is blood agar enriched, selective, or differential?

differential and enriched

blood agar ingredients

sheep’s blood

is macconkey agar enriched, selective, or differential?

selective (enteric gram-negative) and differential (ability to ferment lactose)

macconkey agar ingredients

selective- crystal violet and bile salts

differential- substrate lactose and pH indicator neutral red

is ccna enriched, selective, or differential?

selective (gram positive) and differential (hemolysis)

ccna ingredients

selective- colistin (polymyxin b) and naladixic acid (both antibiotics)

differential- 5% sheep’s blood

is s+s agar enriched, selective, or differential?

selective (enteric gram-negative) and differential (ferment lactose and reduce sulfates)

s+s ingredients

selective- bile salts, brilliant green dye

differential- neutral red pH

is msa enriched, selective, or differential?

selective (halotolerant organisms) and differential (ferment mannitol)

msa ingredients

selective- 7.5% nacl

differential- substrate mannitol (sugar alcohol) and pH indicator phenol red

5 steps for unknown isolation

1. observation of mixed culture by gram staining

2. obtain isolated colonies

3. use colony morphology/gram stain to distinguish your pathogen from your contaminant and identify pure colonies

4. inoculate a stock slant with bacteria from an isolated and pure colony

5. confirm the purity of your stock slants

iodine purpose

mordant

iodine step

2

safranin purpose

counterstain

safranin step

4

crystal violet purpose

primary stain

crystal violet step

1

ethanol purpose

decolorizer

ethanol step

3

heat fix purpose

1. adheres the bacteria to the slide surface

2. kills most microorganisms without destroying structural features

3. enables many stains to better penetrate or react with the microorganisms

heat fix step

6

drying purpose

must be done before heat fixing to prevent the microorganisms from being boiled and destroying structural features needed for staining

drying step

5

both purple and pink stained, with same morphology- pure or mixed?

pure

pure or mixed- both purple and pink stained rods and cocci

mixed

only purple rods- pure or mixed?

pure

gram stain, gram reaction, shape, arrangement

gram positive cocci clusters

gram negative single rods

shape, gram type, arrangement; is it a pure culture?

gram positive single cocci; pure

what aseptic techniques are needed for identifying unknown organism?

why are aseptic techniques important?

identify test. positive or negative? what identification pathway are these tests a part of?

DNAse; negative, positive; gram positive

identify test. positive or negative? what identification pathway are these tests a part of?

identify test. positive or negative? what identification pathway are these tests a part of?

mannitol salt agar (msa); organism can ferment mannitol; gram positive

how to know if a test is catalase positive?

it bubbles

would you use a catalase test on gram negative or gram positive organisms?

gram positive

what does the catalase test stand for?

based on how organisms detoxify hydrogen peroxide; differentiates staphylococcal (+) from streptococcal/enterococcal (-)

what tests would you use for a gram positive, catalase negative organism?

Blood agar, SXT antibiotic, BEA slant, PYR Broth

what test would you use for a gram positive, catalase positive organism?

Mueller Hinton Agar plate; Novobiocin antibiotic disk; DNAse Agar plate; Mannitol Salt Agar plate; PYR broth

what test would you use for a gram negative organism?

Triple-Sugar Iron (TSI) slant; Microgen GNA-ID panel

beta hemolysis

complete lysis- complete clearing of blood around colonies

alpha hemolysis

partial lysis; greenish-gray/yellow around colonies

gamma hemolysis

no hemolysis; no change in medium

identify the hemolysis

what does well 7 test for? how do you interpret the results?

Presence of beta-galactosidase activity; if well turns yellow, there is activity.

Presence of nitrate reductase; red indicates presence of nitrites (positive for nitrate reduction); zinc- red means negative for nitrite reduction, no color- positive for nitrate reduction

if a tsi slant has a black discoloration on the slant, does that mean it is acidic or alkaline?

acidic

interpret the tsi

1. A/A, CO2 -, H2S -

2. A/A, CO2+, H2S -

3. A/A, CO2- , H2S+

4. A/A, CO2+, H2S+

interpret the tsi

1. A/K, CO2-, H2S-

2. A/K, CO2+, H2S-

3. A/K, CO2-, H2S+

4. A/K, CO2+, H2S-

5. K/K, CO2-, H2S-

if your organism produces acid for both the butt and slant on a tsi slant but doesn’t grow pink (lactose fermenting) colonies on mac, why might this occur?

interpret nitrate well

Nitrate reduction negative

which wells on the gram negative test strip require reagents before reading?

8, 10, 12

what pH indicators are used within the different wells?

Bromthymol blue, phenol red,

describe the differences in methods and testing between a kirby-bauer test and an e-test

kirby-bauer test- assess bacterial sensitivity to antibiotic

e-test- determine a specific antibiotics’ minimum inhibitory concentration (MIC)

what is the zone of inhibition for the antibiotic?

36 mm

what is the zone of inhibition for the antibiotic?

31 mm

how would you measure the zones of inhibition for this test? what is this test?

measure diameter when possible, measure radius and double if unable to measure diamter

why would e. coli and s. aureus show different sensitivity to the same beta-lactam antibiotic?

how does the structural difference between gram-positive and gram-negative bacteria affect the zone of inhibition observed with different chemical agents, and why might a chemical be effective for disinfection but not antisepsis?

what is the difference between broad-spectrum and narrow-spectrum drugs?

what is the MIC for the given E-test? ug/mL

what is the MIC for the given E-test? ug/mL

what is the minimal inhibitory concentration on the picture below?

various chemical tests were tested on an organism. which chemical is most effective against the pathogen based on the following zones of inhibition?

·       Chemical 1: 25 mm

·       Chemical 2: 3.6 cm

·       Chemical 3: 0.3 cm

·       Chemical 4: 12 mm

given this disk diffusion assay, rank each chemical in terms of sensitivity.

for the following chemical testing, which of the 3 bacterial species shown are most susceptible to the chemical?

does gram reaction result determine the effectiveness that some chemicals will have against the bacteria? why?

what structures enable gram-negative bacteria to be more resistant, generally, to most chemical agents?

what chemical class impacted your bacteria the most? why do you think that is?

after placing filter disks on an agar plate, what happens to the chemical as it diffuses into the agar?

what are some environmental factors that affect the effectiveness of a chemical?

disinfection

antisepsis

sanitization

why might a chemical be effective for disinfection but not for antisepsis?

hydrogen peroxide class

oxidizing agent

when to use hydrogen peroxide

spores, fungus, strep, etc

ethanol class

when to use ethanol

cavicide class

when to use cavicide

bleach solution class

when to use bleach solution

mouthwash class

when to use mouthwash

what occurs to DNA in the thermocycler?

what is thermocycling and why is it important when running a PCR gel?

name the steps of PCR at numbers 1, 2, and 3 and the temperature that each step occurs at

why might no bands appear after running a PCR? how might this be avoided?

what was placed in the well before running the gel electrophoresis?

what are the approximate sizes of the bands in this gel?

what are the approximate sizes of the bands in this gel?

what gene was amplified for our PCR reaction?

what advantage does 16S rRNA PCR have over traditional biochemical identification methods?

it is faster and more sensitive

thermocycler purpose

SybrSafe DNA dye purpose

16S rRNA primers purpose

why do fermented foods have longer shelf lives and are less prone to spoilage?

what is the first bacterial strain added during fermentation?

what is the second bacterial strain added during fermentation?