Lab Exam 2 Flashcards by Joshua Atallah

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

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Pure Culture Techniques

What is the difference between a broth and agar?

Broth is a liquid media, while agar is a solid media.

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Pure Culture Techniques

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

Flat top (slab)
Angled top (slant)
Petri dish (plates)

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Pure Culture Techniques

What were the two tools used to inoculate new cultures?

Cotton Swab
Inoculating loop

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Pure Culture Techniques

What does TSA and TSB stand for?

TSA = Tryptic Soy Agar
TSB = Tryptic Soy Broth

Tryptic contains amino acids.

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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.

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Pure Culture Techniques

What are examples of nutrients that can be used in complex media?

Egg yolk
Yeast
Soy

TSA/TSB

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Pure Culture Techniques

Is our standard media for our lab complex or defined?

Our media is complex

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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

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Pure Culture Techniques

What have been the two plating techniques introduced in the lab?

Streak plate
Lawn plate

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Pure Culture Techniques

Which plating technique is used to produce pure, isolated colonies from a mixed sample?

Streak plate

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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.

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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

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Effect of Temperature on Growth

How did we apply our microorganism to the surface of this plate?

Used lawn plate technique/ cotton swab

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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

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Effect of Temperature on Growth

Which temperature in degrees celsius, is room temperature?

25 degrees celsius

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Effect of Temperature on Growth

Which temperature in degrees celsius, is body temperature?

37 degrees celsius

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Effect of Temperature on Growth

What temperature in degrees celsius, is refrigerator temperature?

4 degrees celsius

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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

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Effect of Temperature on Growth

What optimal growth group did the temperatures grow best at?

35-45 degrees celsius

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Effect of Temperature on Growth

What GENUS did not grow well at room temperature?

Mycobacterium

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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.

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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

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Evaluation of Antiseptics

How did we apply our microorgansim to the surface of this plate?

Used lawn plate technique/ cotton swab

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# 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?

1. We can look at the data table to see how big or small the death zones are in mm. 2. We can visually see the plate and see if there is a death zone or not | Death zone means that the anitseptic killed bacteria. ## Footnote 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? ## Footnote Should be able to come to proper conclusions if given data table/agar plate

1. We can look at data table to read the death zone in mm 2. 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?

1. Eosin Methylene Blue 2. Mannitol-Salt Agar 3. 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?

1. Eosin Methylene Blue: Dark purple 2. Mannitol-Salt Agar: Red 3. MacConkey Agar: Berry

# Selective and DIfferential Media How is each plate selective? ## Footnote 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? ## Footnote 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.