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?
prevent the spread and contamination of various microbes
what is the difference between a broth and an agar?
broth is liquid growth media
agar is solid growth media
what are the types of agar we used in class?
within a test tube: angled (slant)
in a petri dish: (plate)
what were the two tools that we have used to inoculate new cultures?
loop
cotton swab
what does the abbreviation TSA & TSB stand for?
TSA: Tryptic soy agar
TSB: Tryptic soy broth
what is the difference between a defined and a complex media?
defined: exact chemical composition is known
complex: exact chemical composition varies and is unknown
Give some examples of what can be used as nutrients in a complex medium.
amino acids
glucose
vitamins
nitrogen
Is our standard media for our lab complex or defined?
complex
what have been the 3 plating techniques introduced in lab?
Streak
Lawn
Zag
what are the steps for streak plate?
- 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
what are the steps for lawn plate?
take cotton swab of bacteria and cover entire plate, leaving no gaps
what are the steps for zag plate?
use loop to swab left and right in a few zig-zags
which of the plating techniques is used to produce pure, isolated colonies from a mixed sample?
streak plate
why do we flame the loop between each zone when making a streak plate?
to reduce number of microbes as we progress each step
what bacteria did we use for bacterial tranformation?
Escherichia coli
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
which gene, when expressed, allows the colonies to glow? what does it stand for? come from?
GFP gene
green fluorescence protein
jellyfish
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
what is the purpose of LB in the plates?
to provide microbes with nutrients
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
what plate shows GFP expression?
LB/amp/arabinose plate
In the LB/amp plate, what side (+ or -) should show growth?
the positive side
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 -
how did we apply our microbes in the temperature experiment?
lawn plate - use cotton swab with bacteria to cover entire plate, leaving no gaps
what were all the temperatures used in the temperature experiment?
4
25
37
42
47
60
Which temperature in °C is room temperature?
25 °C
Which temperature in °C is body temperature?
37 °C
What temperatures in °C is refrigerator temperature?
4 °C
Know how to convert temperatures.
°F = (°C x 1.8) + 32
What temperature did all species grow best at?
37 °C
What genus did not grow well at room temperature?
Mycobacterium
Why didn’t the bacteria grow at refrigerator temperature?
the bacteria underwent bacteriostasis in the cold temperature. Bacteriostasis being the slowing of microbial growth
what organisms were used in the antiseptic experiment?
Escherichia coli
Pseudomonas aeruginosa
Bacillus subtilis
Staphylococcus aureus
Mycobacterium smegmatis
How did we apply our microorganism to the surface of the antiseptic experiment?
Lawn plate: take cotton swab with bacteria and cover entire plate, leaving no gaps
What were the chemicals used in antiseptic experiment? what is its general chemical type?
Iodine – halogen
3% Hydrogen peroxide - Peroxygens
2% chlorhexidine - Biguanides
70% isopropyl alcohol – alcohol
1% formaldehyde - Aldehydes
1% silver nitrate - metal
How did we evaluate whether the antiseptic was effective or not?
an antiseptic was effective when its death zone was large
an antiseptic was not effective when its death zone was small/nonexistent
Which organisms were the most and least resistant in the antiseptic experiement?
Most resistant – Escherichia coli
Lest resistant – Mycobacterium smegmatis
which chemicals were most and least effective in the antiseptic experiment?
Most effective – 1% formaldehyde (largest death numbers)
Least effective – 70% isopropyl alcohol
Which organisms were used in the antibiotic/antimicrobial experiment?
Escherichia coli
Pseudomonas aeruginosa
Bacillus subtilis
Staphylococcus aureus
Mycobacterium smegmatis
How did we apply our microorganism to the antibiotic/antimicrobial experiment?
Lawn plate: take cotton swab with bacteria and cover entire plate, leaving no gaps
What were the six antibiotics used for this lab exercise? their mode of action?
Ciprofloxacin – DNA synthesis inhibitors
Chloramphenicol - protein synthesis inhibitor
Gentamicin - protein synthesis inhibitor
Penicillin - cell wall synthesis inhibitor
Colistin - cell wall synthesis inhibitor
Bacitracin - cell wall synthesis inhibitor
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
Which term is used to describe when a bacterium is easily killed by a particular antibiotic?
susceptible
what is narrow spectrum?
targets a single microbe group
what is broad spectrum?
inhibits/kills multiple types of organisms
Which three antibiotics 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 -)
Which organisms were most and least resistant in the antibiotic experiment?
Most resistant: Escherichia coli
Least resistant: Mycobacterium smegmatis
Which antibiotics were most and least effective in the antibiotic experiment?
Most effective: Chloramphenicol
Least effective: Bacitracin
What is the definition of a selective media?
Inhibits growth of unwanted organisms (only the selected microbes live)
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)
What were the three specific plates that we used in our lab? what do the acronyms stand for? what is their original color before inoculation?
(MSA) Mannitol Salt Agar (red)
(EMB) Eosin Methylene Blue (dark purple)
(MAC) MacConkey Agar (raspberry)
Which plate used in lab this semester is not selective or differential?
TSA
Which organisms used in selective/differential experiment are Gram + ?
Staphylococcus aureus
Staphylococcus epidermidis
Which organisms used in selective/differential experiment are Gram - ?
Escherichia coli
Enterobacter aerogenes
Proteus vulgaris
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 +
how is MSA differential?
differential for mannitol fermentation
how is EMB differential?
differential for lactose fermentation
how is MAC differential?
differential for lactose fermentation
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 aerogenes)
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 aerogenes)
nonfermenter: entire plate, including growth, turns brown (Proteus vulgaris)
This is a MAC plate, which genes and species is present?
Escherichia coli
This is a MAC plate, which genes and species is present?
Enterobacter aerogenes
This is a MAC plate, which genes and species is present?
Proteus vulgaris
This is a EMB plate, which genes and species is present?
Escherichia coli
This is a EMB plate, which genes and species is present?
Enterobacter aerogenes
This is a EMB plate, which genes and species is present?
Proteus vulgaris
This is a MSA plate, which genes and species is present?
Staphylococcus aureus
This is a MSA plate, which genes and species is present?
Staphylococcus epidermidis
