Lab Exam 2 Flashcards
Pure Culture Techniques
What is a culture?
What is inoculate?
What is inoculum?
- A culture is growing microbes in a laboratory setting.
- Inoculate: add bacteria to a sample
- Inoculum: Microbes in a growth medium
Pure Culture Techniques
What is the difference between a broth and agar?
Broth is a liquid media, while agar is a solid media.
Pure Culture Techniques
What are the types of agars that we use in class?
- Flat top (slab)
- Angled top (slant)
- Petri dish (plates)
Pure Culture Techniques
What were the two tools used to inoculate new cultures?
- Cotton Swab
- Inoculating loop
Pure Culture Techniques
What does TSA and TSB stand for?
- TSA = Tryptic Soy Agar
- TSB = Tryptic Soy Broth
Tryptic contains amino acids.
Pure Culture Techniques
What is the difference between complex and defined media?
Complex media: We do not know the exact amount of ingredients.
Defined media: We do KNOW the exact the amount of ingredients.
Pure Culture Techniques
What are examples of nutrients that can be used in complex media?
- Egg yolk
- Yeast
- Soy
TSA/TSB
Pure Culture Techniques
Is our standard media for our lab complex or defined?
Our media is complex
Pure Culture Techniques
What is the enrichment medium (even though we didn’t use them in our lab)?
They’re used to grow rare oragnisms. In order to do this we have to make growth conditions specific for the microbe type.
Playing favorites
Pure Culture Techniques
What have been the two plating techniques introduced in the lab?
- Streak plate
- Lawn plate
Pure Culture Techniques
Which plating technique is used to produce pure, isolated colonies from a mixed sample?
Streak plate
Pure Culture Techniques
Why do we flame the loop between each zone when making a streak plate?
We want to reduce the number of bacterial cells. We would like to further dilute the sample.
Effect of Temperature on Growth
What are the organisms used for this exercise?
5 of them
- Mycobacterium smegmatis
- Escherichia coli
- Staphylococcus aurues
- Pseudomonas aeruginosa
- Bacillus subtilis
Effect of Temperature on Growth
How did we apply our microorganism to the surface of this plate?
Used lawn plate technique/ cotton swab
Effect of Temperature on Growth
What are the temperatures that were chosen for this lab?
In celsius
- 4 degrees celsius
- 25 degrees celsius
- 35 degrees celsius
- 37 degrees celsius
- 45 degrees celsius
- 60 degrees celsius
Effect of Temperature on Growth
Which temperature in degrees celsius, is room temperature?
25 degrees celsius
Effect of Temperature on Growth
Which temperature in degrees celsius, is body temperature?
37 degrees celsius
Effect of Temperature on Growth
What temperature in degrees celsius, is refrigerator temperature?
4 degrees celsius
Effect of Temperature on Growth
What is the equation to convert celsius to fahrenheit?
What is the equation to convert fahrenheit to celsius?
- 1.8(X celsius) + 32 = Fahrenheit or (X celsius multiplied by 9/5) + 32 = Fahrenheit
- (X Fahrenheit - 32) x 5/9 = Celsius
Effect of Temperature on Growth
What optimal growth group did the temperatures grow best at?
35-45 degrees celsius
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 well at refrigerator temperature?
Because a refrigerator is at 4 degrees celsius. At this temp, it has a bacteriostasis effect on bacteria. This does not allow them to grow.
Evaluation of Antiseptics
Which organisms were used in this lab exercise?
The same 5 as the “Effects of Temperature”
- Mycobacterium smegmatis
- Escherichia coli
- Staphylococcus aureus
- Pseudomonas aeruginosa
- Bacillus subtilis
Evaluation of Antiseptics
How did we apply our microorgansim to the surface of this plate?
Used lawn plate technique/ cotton swab
Evaluation of Antiseptics
What were the chemicals used in lab?
6 of them
- Iodine
- Hydrogen peroxide
- Chlorhexidine
- Isopropyl alcohol
- Formaldehyde
- Silver nitrate
Evaluation of Antiseptics
How did we evaluate whether the antiseptic was effective or not?
- We can look at the data table to see how big or small the death zones are in mm.
- We can visually see the plate and see if there is a death zone or not
Death zone means that the anitseptic killed bacteria.
Big death zone = susceptible
Small death zone = resistant
Evaluation of Antiseptics
Which organisms were the most and least resistant?
- Most resistant: Escherichia coli
- Least resistant: Mycobacterium smegmatis
Evaluation of Antiseptics
Which chemcials were most and least effective?
- Most effective: Formaldehyde
- Least effective: Isopropyl alcohol
Evaluation of Antiseptics
Each antiseptic has a chemical type, what are they?
- Iodine = Halogen
- Hydrogen peroxide = Oxidizing agent
- Chlorohexidine = Biguanide
- Isopropyl alcohol = Alcohol
- Formaldehyde = Aldehyde
- Silver nitrate = Heavy metal
Evaluation of antibiotics
Which organisms were used in this lab exercise?
Same 5 as “Effects on temperature” and “Evaluation of antiseptics”
- Mycobacterium smegmatis
- Escherichia coli
- Staphylococcus aureus
- Pseudomonas aerugniosa
- Bacillus subtillis
Evaluation of antibiotics
How did we apply our microorganism to the surface of this plate?
Lawn plate technique/ cotton swab
Evaluation of antibiotics
What were the six antibiotics used in this lab exercise?
- Chloramphenicol
- Cirprofloxacin
- Gentamicin
- Penicillin
- Colistin
- Bacitracin
Evaluation of antibiotics
What are the basic modes of action for each antibiotic that was used?
6 of them
- Chloramphenicol = Inhibiting protein synthesis
- Cirprofloxacin = Nucleic acid inhibitor
- Gentamicin = Inhibiting protein synthesis
- Penicillin = Inhibiting cell wall synthesis
- Colistin = Inhibiting cell wall synthesis
- Bacitracin = Inhibiting cell wall synthesis
Evaluation of antibiotics
How did we evaluate whether the antibiotic was effective or not?
Should be able to come to proper conclusions if given data table/agar plate
- We can look at data table to read the death zone in mm
- We can visually see the agar plate and spot the death zone
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 selectively kill Gram + bacteria or Gram - bacteria?
- Penicillin did not kill a few of the bacteria in the experiment. We know this because we don’t see a death zone for two of the bacteria (E. Coli & P. aeruginosa are gram -). This means that they selectively kill gram + bacteria.
- Colistin did not kill a few of the bacteria in the experiment. We know this because we don’t see death zone for two of the bacteria (B. subtillis & S. aureus are gram +). This means they selectively kill gram - bacteria.
Evaluation of antibiotics
Which organisims were most and least resistant?
- Most resistant = Escherichia coli
- Least resistant = Mycobacterium smegmatis
Evaluation of antibiotics
Which antibiotics were most and least effective?
- Most effective: Chloramphenicol
- Least effective: Colistin
Bacterial Transformation Lab
What bacteria did we use for this lab?
Escherichia Coli
Bacterial Transformation Lab
What genes are found on the pGLO plasmid?
- B-lactamase gene
- GFP gene
- araC gene
Bacterial Transformation Lab
Which gene, when expressed, allows the colonies to glow?
GFP
Bacterial Transformation Lab
What is the purpose of the LB in the plates?
To grow bacteria and provide nutrients
Bacterial Transformation Lab
What inoculated plates or side of plates showed transformation?
- LB/AMP+
- LB/AMP/ARA
“ARA” because of arabinose
Bacterial Transformation Lab
What is needed in the media for the colonies to glow?
Arabinose? I’m unsure of this answer.
Bacterial Transformation Lab
What plate shows GFP expression?
LB/AMP/ARA
Bacterial Transformation Lab
In the LB/AMP plate, what side (+ or -) should show growth?
In the LB/AMP plate, the + side should show growth.
Unsure about this answer, ask for help.
Bacterial Transformation Lab
What is the name of the antibiotic resistance gene? What antibiotic is it resistant?
- B-Lactamase
- It is resistant against the amipicillin antibiotic.
Bacterial Transformation Lab
What is transformation?
The bacterial cells ability to uptake naked DNA.
Bacterial Transformation Lab
What are competent cells?
Competent cells are bacteria that can uptake naked DNA naturally. An example of this is Bacillus Subtilis.
Escherichia Coli is not naturally competent. Must be made competent.
Bacterial Transformation Lab
How did we make the bacteria competent in the lab?
Cells were treated with calcium chloride and subjected to heat shock.
Bacterial Transformation Lab
What is a plasmid?
Circular DNA found in bacteria cells.
Bacterial Transformation Lab
What organism did GFP originate from?
GFP originates from marine jellyfish
Selective and DIfferential Media
What is the definition of a selective media?
Inhibits certain bacteria while others survive.
Selective and DIfferential Media
What is the definition of differential media?
Bacteria will grow but look different (color change).
Selective and DIfferential Media
What were the three specific plates that were used in our lab?
- Eosin Methylene Blue
- Mannitol-Salt Agar
- MacConkey Agar
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.
Hektoen Enteric plate
Selective and DIfferential Media
Which organisms used in this exercise are Gram + ?
Staphylococcus auerus, Staphylococcus epidermidis
Selective and DIfferential Media
Which organisms used in this exercise are Gram - ?
Escherichia coli, Enterobacter aerogenes, Proteus vulgaris
Selective and DIfferential Media
What are the original colors of each type of plate before the experiment starts?
- Eosin Methylene Blue: Dark purple
- Mannitol-Salt Agar: Red
- MacConkey Agar: Berry
Selective and DIfferential Media
How is each plate selective?
MSA, EMB, MAC
- Mannitol Salt agar selects for Gram +, it is selective against Gram -
- Eosin Methylene Blue and MacConkey agar selects for Gram -, it is selective against Gram +
Selective and DIfferential Media
How is each plate differential?
MSA, EMB, MAC
- Mannitol-Salt agar is differential based on mannitol fermentation.
- Eosin Methylene Blue & MacConkey agar are differential based on lactose fermentation.
Selective and DIfferential Media
Visually identify the results for the lab exam.