Lab Exam 2 Flashcards
What is a culture?
growing bacteria in a lab setting
What is an inoculum?
placing bacteria in a growth medium
- Why do we use aseptic technique in lab?
to prevent cross contamination
the difference between a broth and an agar
broth is liquid growth media
agar is solid growth media
What are the types of agars that we use in class?
within a test tube: angled (slant)
in a petri dish: (plate)
What were the three tools that we have used to inoculate new cultures?
loop
cotton swab
What does the abbreviation TSA & TSB stand for?
Tryptic soy Agar
Tryptic soy broth
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
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
Is our standard media for our lab complex or defined?
complex
You should know the scientific foundation of an enrichment medium even though we do not use them in our lab
what have been the 3 plating techniques introduced in lab?
Lawn, zigzag, streak
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
Steps for zigzag:
use loop to swab left and right in a few zig-zags
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
Which of the plating techniques is used to produce pure, isolated colonies from a mixed sample?
Streak plating
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
Bacterial Transformation Lab
what bacteria did we use for bacterial transformation?
Escherichia coli
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
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
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
Bacterial Transformation Lab
what is the purpose of LB in the plates?
to provide microbes with nutrients
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
Bacterial Transformation Lab
what plate shows GFP expression?
LB/amp/arabinose plate
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)
