Lab Exam 2 Flashcards by Cameron Burtt

What is a culture?

growing bacteria in a lab setting

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What is an inoculum?

placing bacteria in a growth medium

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Why do we use aseptic technique in lab?

to prevent cross contamination

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the difference between a broth and an agar

broth is liquid growth media
agar is solid growth media

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What are the types of agars that we use in class?

within a test tube: angled (slant)
in a petri dish: (plate)

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What were the three tools that we have used to inoculate new cultures?

loop
cotton swab

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What does the abbreviation TSA & TSB stand for?

Tryptic soy Agar

Tryptic soy broth

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What is the difference between a complex and a defined media

Complex media = Exact contects are unknown

Defined media = Every ingredient is know in its exact amount

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Give some examples of what can be used as nutrients in a complex medium

Extracts from eggs, years, soy, animals

amino acids
glucose
vitamins
nitrogen

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Is our standard media for our lab complex or defined?

complex

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You should know the scientific foundation of an enrichment medium even though we do not use them in our lab

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what have been the 3 plating techniques introduced in lab?

Lawn, zigzag, streak

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Steps for streak plating:

spread one loopful of bacteria on 1/4 of plate
flame loop
start in zone 1 and spread bacteria over 1/4 of plate
flame loop
start in zone 2 and spread bacteria over 1/4 of plate
flame loop
start in zone 3 and spread bacteria over 1/4 of plate, avoiding zone 1 & 2

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Steps for zigzag:

use loop to swab left and right in a few zig-zags

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Steps for lawn plating:

used a sterile cotton swab and aseptic technique to apply bacteria to the lawn plate
Smeared the entire surface, leaving no gaps

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Which of the plating techniques is used to produce pure, isolated colonies from a mixed sample?

Streak plating

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Why do we flame the loop between each zone when making a streak plate?

To prevent build up because you want to reduce bacterial numbers

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Bacterial Transformation Lab

what bacteria did we use for bacterial transformation?

Escherichia coli

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Bacterial Transformation Lab

What genes are found on the pGLO plasmid? what do they code for?

bla gene: allows resistance to ampicillin
araC gene: blocks GFP gene from being able to express (glow) all the time
GFP gene: codes for fluorescence

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Bacterial Transformation Lab

which gene, when expressed, allows the colonies to glow? what does it stand for? come from?

GFP gene
green fluorescence protein
jellyfish

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Bacterial Transformation Lab

what is required in media in order for colonies to glow?

arabinose in the plate allows GFP gene to be expressed (glow) by blocking the araC gene from blocking GFP gene

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Bacterial Transformation Lab

what is the purpose of LB in the plates?

to provide microbes with nutrients

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Bacterial Transformation Lab

what inoculated plates or side of plates showed transformation? why?

LB/amp (+pGLO side) - because growth was present due to bla gene being present within the plasmid

LB/amp/arabinose plate - because growth was present and it fluoresced

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Bacterial Transformation Lab

what plate shows GFP expression?

LB/amp/arabinose plate

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Bacterial Transformation Lab In the LB/amp plate, what side (+ or -) should show growth?

the positive side

Bacterial Transformation Lab what is the name of the antibiotic resistance gene? what antibiotic is it resistant towards?

bla gene - resistant to ampicillin

Gram + or gram - : Bacillus subtilis?

gram +

Gram + or gram - : Escherichia coli?

gram -

Gram + or gram - : Staphylococcus aureus?

gram +

Gram + or gram - : Mycobacterium smegmatis?

acid-fast gram +

Gram + or gram - : Pseudomonas aeruginosa?

gram -

Effect of Temperature on Growth You should know the list of organisms used for this exercise

Bacillus Escherichia Mycobacterium Psedomonas

Effect of Temperature on Growth How did we apply our microorganism to the surface of this plate? (Know the name of the plating method and be able to describe the method.

lawn plate - use cotton swab with bacteria to cover entire plate, leaving no gaps

Effect of Temperature on Growth You should know the temperatures that were chosen for this lab exercise.

4 °C 25°C (room temp 37°C 42°C 47°C 52°C

Effect of Temperature on Growth Which temperature in °C is room temperature?

25 °C

Effect of Temperature on Growth Which temperature in °C is body temperature?

37 °C

Effect of Temperature on Growth What temperatures in °C is refrigerator temperature?

4 °C

Effect of Temperature on Growth Know how to convert temperatures.

ferinheit = (Celsius x 1.8) + 32 Celsius = ferinheit - 32 / 1.8

Effect of Temperature on Growth What temperature did all species grow best at?

37°C

Effect of Temperature on Growth -What genus did not grow well at room temperature?

Mycobacterium

Effect of Temperature on Growth Why didn’t the bacteria grow at refrigerator temperature?

Bacteriostasis slows down / inhibits the growth

Evaluation of Antiseptics Which organisms were used in this lab exercise?

Escherichia Coli Psudomonas aeruginosa Bacillus subtilis Staohylococcus aureus Mycobacterium smegmatis

Evaluation of Antiseptics How did we apply our microorganism to the surface of this plate? (Know the name of the technique and be able to describe the technique.

Lawn plate: take cotton swab with bacteria and cover entire plate, leaving no gaps

Evaluation of Antiseptics What were the chemicals used in lab?

Iodine – halogen 3% Hydrogen peroxide - Peroxygens 2% chlorhexidine - Biguanides 70% isopropyl alcohol – alcohol 1% formaldehyde - Aldehydes 1% silver nitrate - metal

Evaluation of Antiseptics How did we evaluate whether the antiseptic was effective or not? You should be able to come to proper conclusions if I provide you with a data table or agar plates.

Test microbe in many antiseptics: - most effective would have the largest death zone - least effectove had small to no death zone

Evaluation of Antiseptics You should know the overall data trend from the project.

Evaluation of Antiseptics Which organisms were the least resistant?

Mycobacterium smegmatis

Evaluation of Antiseptics Whic organisim was the most resistant?

Escherichia coli

Evaluation of Antiseptics Which chemicals were least effective?

Isopropyl alcohol

Evaluation of Antiseptics Which chemicals were most effective?

Formaldehyde

Evaluation of Antiseptics You should be able to group each antiseptic into its general chemical type, such as metal, alcohol, etc. This matches chapter 7 from lecture. (Look up each antiseptic type in Chapter 7)

Evaluation of Antibiotics Which organisms were used in the antibiotic/antimicrobial experiment?

Escherichia coli Pseudomonas aeruginosa Bacillus subtilis Staphylococcus aureus Mycobacterium smegmatis

Evaluation of Antibiotics How did we apply our microorganism to the surface of this plate? (Know the name of the technique and be able to describe the technique.)

Lawn plate: take cotton swab with bacteria and cover entire plate, leaving no gaps

Evaluation of Antibiotics What were the six antibiotics used for this lab exercise?

Chloramphenicol - protein synthesis inhibitor Ciprofloxacin – DNA synthesis inhibitors Gentamicin - protein synthesis inhibitor Penicillin - cell wall synthesis inhibitor Colistin - cell wall synthesis inhibitor Bacitracin - cell wall synthesis inhibitor

Evaluation of Antibiotics How did we evaluate whether the antibiotic was effective or not?

Larger death zone meant the antibiotic was effective Small death zone meant the antibiotic was ineffective

Evaluation of Antibiotics Which term is used to describe when a bacterium is easily killed by a particular antibiotic?

susceptible

Evaluation of Antibiotics what is narrow spectrum?

targets a single microbe group

Evaluation of Antibiotics what is broad spectrum?

inhibits/kills multiple types of organisms

Evaluation of Antibiotics Which antibiotic did not kill a few of the bacteria used in the experiment? How do we know that it did not kill? Does this antibiotic selectively kill Gram + bacteria or Gram - bacteria?

Penicillin did not kill Pseudomonas aeruginosa or Mycobacterium smegmatis because there was no death zone present. Penicillin is narrow spectrum for Gram + (does not target gram -) Colistin did not kill Bacillus subtilis or Staphylococcus aureus because there was no death zone present. Colistin is narrow spectrum for Gram - (does not target gram +) Bacitracin did not kill Escherichia coli nor Pseudomonas aeruginosa because there was no death zone present. Bacitracin is narrow spectrum for Gram + (does not target gram -)

Evaluation of Antibiotics -Which antibiotics were most and least effective?

Most effective: Chloramphenicol Least effective: Bacitracin

Which organisms were most and least resistant in the antibiotic experiment?

Most resistant: Escherichia coli Least resistant: Mycobacterium smegmatis

Selective and Differential Media What is the definition of a selective media?

Inhibits growth of unwanted organisms (only the selected microbes live)

Selective and Differential Media - What is the definition of a differential media?

Allows organisms to grow but the added chemicals make them look different (they change different colors and/or precipitate is visible)

Selective and Differential Media What were the three specific plates that we used in our lab? What color are they?

(MSA) Mannitol Salt Agar (red) (EMB) Eosin Methylene Blue (dark purple) (MAC) MacConkey Agar (raspberry)

Selective and Differential Media Which plate used in lab this semester is not selective or differential? This plate would not be part of the S&D experiment.

TSA

Selective and Differential Media Which organisms used in this exercise are Gram +?

Staphylococcus aureus Staphylococcus epidermidis

Selective and Differential Media Which organisms used in this exercise are Gram -?

Escherichia coli Enterobacter aerogenes Proteus vulgaris

What does EMB stand for?

Eosin Methylene Blue

What does MAC stand for?

MacConkey Agar

What does MSA stand for?

Mannitol Salt Agar

Selective and Differential Media how is MSA selective?

selects for gram + selects against gram -

how is EMB selective?

selects for gram - selects against gram +

how is MAC selective?

selects for gram - selects against gram +

Selective and Differential Media how is MSA differential?

differential for mannitol fermentation

how is EMB differential?

differential for lactose fermentation

how is MAC differential?

differential for lactose fermentation

Selective and Differential Media what type of fermenter and what are the color possibilities for MSA? what genes and species would be responsible for each?

fermenter: turns yellow (Staphylococcus aureus) nonfermenter remains red (Staphylococcus epidermidis

what type of fermenter and what are the color possibilities for EMB? what genes and species would be responsible for each?

strong fermenter: turns shiny metallic green (Escherichia coli) weak fermenter: pink around edges of colonies (Enterobacter aeruginosa) nonfermenter: remains dark purple (Proteus vulgaris)

what type of fermenter and what are the color possibilities for MAC? what genes and species would be responsible for each?

strong fermenter: turns bright pink (Escherichia coli) weak fermenter: turns light pink (Enterobacter aeruginosa) nonfermenter: entire plate, including growth, turns brown (Proteus vulgaris)