Micro lab exam 2

what is a culture?

microorganisms that grow and multiply in a prepared nutrient called a culture medium, enabling experimental observation

what is inoculum?

microbes that grow in a medium

• inoculating = putting bacteria in a medium

in pure culture technique, why do we use aseptic technique in lab?

To prevent unwanted contamination of the culture and surroundings.

What is the difference between broth and agar?

Broth is liquid growth medium. Agar is a solid media.

In pure culture technique, what type of agar did we use in class?

Tryptic Soy Agar (TSA)

How do you visually identify a plate, broth, and slant?

Plate: solid agar in a Petri dish

Broth: liquid in a tube

Slant: agar solidified at an angle in a tube.

What two tools did we use to inoculate new cultures?

Inoculating loop and sterile cotton swab

What do TSA and TSB stand for?

Tryptic Soy Agar and Tryptic Soy Broth

What is the difference between complex and defined media?

Complex: exact chemical composition is not fully known

Defined: all chemicals and their amounts are known

What are examples of nutrients used in complex media?

yeast extracts, meat extracts, plant extract, protein digests

In pure culture technique, is our standard growth medium complex or defined?

Complex. TSA and TSB are complex media

What three plating techniques were introduced in lab?

1. Streak plate

2. lawn plate

3. zag plating

What are the steps for streak plating technique?

1. Take 1 loopful of bacteria and spread it over ¼ of the plate in a continuous motion.

2. Flame the loop to reduce numbers of bacteria.

3. From Zone 1, overlap and spread bacteria over the next ¼ of the plate.

4. Flame the loop.

5. Pull from Zone 2, overlap and spread over the next ¼ of the plate.

6. Flame loop.

7. Pull from Zone 3 → DO NOT overlap with Zone 1 or Zone 2.

• Label bottom of plate:

Name

Streak plate #1

• Purpose: Pure culture technique → diluting sample to get complete separate colonies (pure).
  
  

What are the steps for lawn plating technique?

using a sterile cotton swab, cover entire agar surface with bacteria, leaving no gaps

What are the steps for zag plating technique?

streak the surface in a continuous back-and-forth pattern

Which technique produces pure, isolated colonies from a mixed sample?

Streak plate method

In pure culture technique, why do we flame the loop between streak plate zones?

To reduce the number of bacteria transferred, allowing isolated colonies to form

Which organisms were used in our In effect of temperature on growth lab experiment?

1. Escherichia coli

2. Mycobacterium smegmatis

3. Bacillus spizizenii

4. Staphylococcus aureus

5. Pseudomonas aeruginosa

In effect of temperature on growth lab, how did we apply microorganisms to the temperature plates? Name and describe the technique.

Lawn plating: spread bacteria evenly over the agar surface using a sterile swab (leaving no gaps)

What were the six temperatures were chosen for our effect of temperature on growth lab experiment?

1.) 4°C

2.) 25°C

3.) 37°C

4.) 45°C

5.) 52°C

6.) 60°C

What is room temperature in °C?

25°C

What is body temperature in °C?

37°C

What is refrigerator temperature in °C?

4°C

How do you convert temperatures between Celsius and Fahrenheit?

°F = (°C × 9/5) + 32

°C = (°F − 32) × 5/9

Which optimal growth group did our bacteria grow best at?

Mesophiles, bacteria that grow best at moderate temperatures, including near 37°C

In effect of temperature on growth lab, what genus did not grow well at room temperature?

Mycobacterium

In effect of temperature on growth lab, why didn't bacteria grow at refrigerator temperature?

Low temperature slowed metabolism and reproduction, causing bacteriostasis

Which organisms were used in the antiseptic experiment?

1. Escherichia coli

2. Mycobacterium smegmatis

3. Bacillus spizizenii

4. Staphylococcus aureus

5. Pseudomonas aeruginosa

In the evaluation of antiseptics lab, how did we apply microorganisms to the antiseptic plates? Name and describe the technique.

Lawn plating: use a sterile swab to spread the bacterial sample evenly over the agar surface, creating a continuous lawn.

What were the six chemicals were used in the antiseptic experiment?

1.) 4% chlorhexidine

2.) 3% hydrogen peroxide

3.) 10% povidone-iodine

4.) 70% isopropyl alcohol

5.) 1% formaldehyde

6.) 1% silver nitrate

In the evaluation of antiseptics lab, how did we evaluate antiseptic effectiveness using plates or data?

Measure the clear zone of inhibition surrounding each antiseptic disk in millimeters.

A larger zone indicates more visible growth inhibition.

A zero indicates no measurable inhibition.

Which organisms were most and least resistant to antiseptics in our experiment?

most resistant = E. coli (25 mm total)

least resistant = M. smegmatis (57 mm total)

Smaller total zones indicate greater resistance

Which antiseptics were most and least effective overall?

most effective = 1% formaldehyde (48 mm total)

least effective = 70% isopropyl alcohol (0 mm total)

What general chemical type does each antiseptic belong to?

Chlorhexidine — biguanide

hydrogen peroxide — peroxygen

povidone-iodine — halogen

isopropyl alcohol — alcohol

formaldehyde — aldehyde

silver nitrate — inorganic salt

Which organisms were used in the antibiotic experiment?

1. Escherichia coli

2. Mycobacterium smegmatis

3. Bacillus spizizenii

4. Staphylococcus aureus

5. Pseudomonas aeruginosa

In the Evaluation of Antibiotics lab, how did we apply microorganisms to the antibiotic plates? Name and describe the technique.

Lawn plating: spread the bacterial sample evenly over the agar surface using a sterile swab, then place antibiotic disks on the inoculated agar.

What six antibiotics were used in the experiment ‘Evaluation of Antibiotics’ ?

1. Chloramphenicol

2. Ciprofloxacin

3. Gentamicin

4. Penicillin

5. Colistin

6. Bacitracin

In the evaluation of antibiotics lab, what is the basic mode of action of each antibiotic?

Chloramphenicol — inhibits protein synthesis

Ciprofloxacin — inhibits DNA replication.

Gentamicin — inhibits protein synthesis

Penicillin — inhibits cell wall synthesis.

Colistin — disrupts bacterial membranes.

Bacitracin — inhibits cell wall synthesis.

How did we evaluate antibiotic effectiveness using plates or data?

Measure the zone of inhibition around each antibiotic disk.

A larger zone means more visible inhibition in this experiment; no zone means no measurable inhibition.

What term describes a bacterium easily inhibited by an antibiotic?

Susceptible (sensitive)

In the evaluation of antibiotics lab, which TWO antibiotics did not inhibit several bacteria? How do we know, and does it mainly affect Gram-positive or Gram-negative bacteria?

Penicillin showed 0 mm zones of inhibition against Escherichia coli (-) , Pseudomonas aeruginosa (-) , and Mycobacterium smegmatis (+,-) , indicating no visible inhibition.

Bacitracin showed 0 mm zones of inhibition against Escherichia coli (-) , Pseudomonas aeruginosa (-) , and Bacillus spizizenii (+) , indicating no visible inhibition.

How do we know? : Penicillin and Bacitracin showed 0mm zones of inhibition against several bacteria, meaning the antibiotic did not prevent bacterial growth.

Do they mainly affect gram positive or gram negative bacteria? :

Both antibiotics mainly affected Gram-positive bacteria, as shown by their zones of inhibition against certain Gram-positive organisms in our experiment. Neither inhibited Gram negative instead.

In the evaluation of antibiotics lab, which organisms were most and least resistant to antibiotics overall?

Most resistant = Pseudomonas aeruginosa (22 mm total)

Least resistant = Mycobacterium smegmatis (64 mm total)

Which antibiotics were most and least effective overall?

Most effective = ciprofloxacin (68 mm total).

Least effective = colistin (15 mm total).

What bacterium did we use for transformation lab?

 Escherichia coli

In bacterial transformation lab, what genes are found on the pGLO plasmid?

1. GFP: Codes for green fluorescent protein, allowing bacteria to fluoresce green when expressed.

2. bla: Codes for beta-lactamase, providing resistance to ampicillin.

3. araC: Codes for a regulatory protein that controls GFP expression in response to arabinose.

In bacterial transformation lab, which gene, when expressed, allows colonies to glow?

GFP, which produces green fluorescent protein.

In bacterial transformation lab, what is the purpose of LB in the plates?

LB provides nutrients needed for bacterial growth.

In bacterial transformation lab, which inoculated plates or sides showed transformation?

+pGLO sides had bacteria growing on LB/amp

and LB/amp/ara (only this one glowed)

they demonstrated successful transformation because they acquired the plasmid and its ampicillin-resistance gene.

In bacterial transformation lab, what is needed in the medium for colonies to glow?

Arabinose, which activates GFP expression in bacteria containing pGLO

In bacterial transformation lab, which plate shows GFP expression?

 +pGLO LB/amp/ara; colonies fluoresce green under UV light

In bacterial transformation lab, in the LB/amp plate, which side (+ or −) should show growth?

+pGLO grows because it has ampicillin resistance; −pGLO does not grow.

In bacterial transformation lab, what is the antibiotic-resistance gene, and which antibiotic does it resist?

bla, which produces beta-lactamase and provides resistance to ampicillin

Transformation lab: Define transformation

Uptake of external DNA by a bacterial cell.

Transformation: Define competent cells

Cells capable of taking up DNA from their surroundings.

How did we make the bacteria competent in our transformation lab? (Four steps)

1. Treated E. coli with calcium chloride transformation solution

2. kept the cells on ice

3. applied a brief heat shock at 42°C

4. followed by cooling

Transformation: Define plasmid

 A small, usually circular DNA molecule separate from the bacterial chromosome that can replicate independently.

In bacterial transformation lab, what organism did GFP originate from?

Aequorea Victoria, a jellyfish species from which green fluorescent protein (GFP) was originally isolated

In selective and differential, what is differential media?

a culture media that allow microorganisms to grow but distinguish them based on visible differences in appearance

ex. color, texture

In selective and differential, what is selective media?

a media inhibits the growth of unwanted microorganisms while allowing desired microorganisms to grow

What three specific plates were used in our selective and differential experiment?

1. EMB (Eosin Methylene Blue Agar)

2. MAC (MacConkey Agar)

3. MSA (Mannitol Salt Agar)

Which plate medium used this semester is neither selective nor differential?

TSA (Tryptic Soy Agar), a general-purpose growth medium

Which organisms in our selective and differential experiment are Gram-positive?

Staphylococcus aureus and Staphylococcus epidermidis

Which organisms in our selective and differential experiment are Gram-negative?

Escherichia coli, Klebsiella aerogenes, and Pseudomonas aeruginosa

What are the genus and species of every organism used in our selective and differential experiment?

1. Staphylococcus aureus

2. Staphylococcus epidermidis

3. Escherichia coli

4. Klebsiella aerogenes

5. Pseudomonas aeruginosa

What are the full names of EMB, MAC, and MSA?

EMB = Eosin Methylene Blue Agar

MAC = MacConkey Agar

MSA = Mannitol Salt Agar

in selective and differential lab, what were the original colors of the three plates before inoculation?

EMB = dark purple

MAC = berry

MSA = red

In selective and differential media, how is each plate selective?

MSA- selects for gram positive, selecting against gram negative

EMB- selects gram negative, selecting against gram positive

MAC- selects for gram negative, selecting against gram positive

How is each plate differential? Explain why colors change, the possible colors, and each organism's expected result.

MSA — Mannitol fermentation

• Staphylococcus aureus: Fermenter → plate turns yellow

• Staphylococcus epidermidis: Non-fermenter → plate remains red

• Why? Mannitol fermentation produces acid, turning phenol red yellow.

EMB — Lactose fermentation

• Escherichia coli: Strong fermenter → shiny metallic green

• Klebsiella aerogenes: Weaker fermenter → pink colonies/ pink around edges

• Pseudomonas aeruginosa: Non-fermenter → colonies remain dark purple

• Why? Acid from lactose fermentation changes the appearance of the dyes.

MAC — Lactose fermentation

• Escherichia coli: Strong fermenter → bright pink color change

• Klebsiella aerogenes: Weaker fermenter → colonies are light pink/puffy colonies

• Pseudomonas aeruginosa: Nonfermenter → colonies match the plate color (more brown than berry after incubation)

• Why? Acid from lactose fermentation changes the neutral red indicator.