Lab exam Flashcards
Where are agar plates discarded?
In the autoclave bucket at the front of the lab
Where are culture tubes discarded?
They are placed in the test tube rack at the front of the lab
Where are microscope slides discarded?
In the used slide container
Where are all broken, but clean glassware and plastic placed?
In the blue bucket at the front of the lab
Where are all broken, contaminated glassware and plastic placed?
In the yellow bucket at the front of the lab.
Where are micropipette tips ejected?
Directly into the biohazard disposal bag located on the bench.
How are culture spills cleaned?
Cleaned up immediately by covering with paper towel and pouring disinfectant onto the covered spill
Assume that you are always working with ____ microorganisms in the lab
BSL2
Microscope dust cover
Cover you put on top
Microscope objective lens
The actual lens that is engaged
Microscope stage
Thing that you put the specimen on
Microscope condenser lens (what it is and function)
First lever under stage, gathers and focuses light from an illuminator, creating a cone of light that illuminates the specimen evenly
Microscope iris diaphragm (what it is and function)
Second lever under stage, controls the amount and angle of light reaching the specimen, influencing brightness, contrast, and depth of field
What is the diopter adjustment ring?
Ring that you can turn on eyepiece to reduce eye strain
What does it mean that the microscope is parfocal?
The objective lenses are parfocal, meaning that once the 10x objective is focused, the 40x or 100x objective can be engaged and focused with only slight manipulation of the fine adjustment knob (this is such bs)
100 on the blue pipette means…
1000 µL, aka 1 mL
100 on yellow pipette means…
100 µL
What 3 things should be included in a scaled cell morphology diagram?
- Figure number/caption
- Total magnification of specimen (multiply power of ocular lens, 10x, by the power of the objective lens from which you made your diagram)
- Magnification of your diagram (divide the length of your diagram by the actual length of the specimen)
What do you need to include in the figure caption of a scaled diagram of a cell? (5)
Figure, what is known about the specimen, properly identify the cell shape, correct total magnification and correct magnification of drawing
If the length of a 5 µm cell drawn in 73 mm, what is the magnification of the drawing?
73 000 µm/5 µm = 14 600x
How many µm is an ocular division in the 10x objective?
10
How many µm is an ocular division in the 40x objective?
2.5
How many µm is an ocular division in the 100x objective?
1
What 4 things should be on a cultured plate label?
Date, lab section, treatment, name
What type of medium contains all essential nutrients for most microorganisms?
Nutrient agar
What is chemically defined media composed of? What does it supply organisms with?
Basal (basic) salt solution, which supplies the inorganic requirements of the organism, and organic components that provide a source of carbon and energy. The exact chemical composition of each component IS known.
What is complex media composed of? What does it supply organisms with?
Complex media contains substances that are rich in both inorganic and organic nutrients such as meat or vegetable infusions, blood and hormones. The exact chemical composition of the media IS NOT known.
What is enriched media composed of? What does it supply organisms with?
Loaded with nutrients and macromolecular monomers to promote the growth of even the most FASTIDIOUS organisms
What is minimal media composed of? What does it supply organisms with?
Contains only the most basic nutrients
What allows microbial cells to grow on agar media?
They are immobilized, which allows them to grow in aggregates of cells that eventually form visible colonies
When one colony morphology (and therefore only one species) is present on an agar plate, we say we have a ____ culture
Pure
What 6 things should you include in colony morphology?
- Shape (form)
- Surface
- Elevation
- Size (in mm)
- Pigment (non-pigmented vs. pigmented, water-soluble pigments diffuse into the media)
- Opacity
Also record any changes to the colour of the media
What are the 5 possibilities for shape colony morphology?
- Punctiform (very small circles)
- Circular
- Irregular (looks like a splatter)
- Filamentous (looks like a spider web)
- Rhizoid (looks like a spider web with fewer branches)
What are the 5 possibilities for surface colony morphology?
- Smooth
- Rough
- Mucoid (looks wet)
- Moist
- Dry
What are the 5 possibilities for elevation colony morphology?
- Flat
- Raised
- Convex (like a mountain)
- Umbonate (raised like a nipple)
- Crateriform (goes into agar)
Nutrient agar (NA) is an example of ___ media
Complex
Minimal glucose is an example of ___ media
Chemically defined
What is an aliquot?
The desired volume of liquid (that is being pipetted)
What 4 things should be included in the cellular morphology of bacteria?
- Cell shape (cocci or bacilli)
- Cell size (recorded as the diameter for cocci and the length for bacilli)
- Cell arrangement
- Gram reaction
What are the 5 possible cell arrangements for cell morphology?
Random (usually what we saw in this course), pairs, tetrads (looks like 4 cells together) chains, clusters (a bunch of cells clumped together intermittently)
What are the 6 steps for preparing a gram stain (including the preparation of a smear)?
- Sterilize the loop by passing it through a flame. Add a drop of water onto the glass slide with a bit of culture and spread it around. Allow to air dry
- Add methanol to fix the cells. Allow to air dry
- Flood the smear with crystal violet for 1 MINUTE. Rinse with water to remove excess stain.
- Flood the smear with Lugol’s iodine for 1 MINUTE. Rinse with water and remove excess water.
- Decolourize with 95% ethanol (drip down the slide alternating with water until the drippings are colourless)
- Counterstain with safranin for 1 MINUTE
How to calculate the number of cells/mL on a plate
Plate count x dilution factor (x conversion factor if less than 1 mL was plated)
If you count 185 colonies on a 10^-3 diluted plate, what would be the concentration of cells on the plate? (assuming 1 mL was plated)
185 x 10^3 = 185,000 cells/mL
If you count 185 colonies on a 10^-4 plate after plating 100µL of the diluted solution onto the plate, how would you calculate the CFU/mL?
185 x 10^4 x 10
First calculate 185 x 10^4=1,850,000 CFU/100 µL
then do:
1,850,000/100 µL x 1000 µL/1 mL
= 1.85 x 10^7
The spread plate that produces a colony count between __ and __ is chosen to perform the CFU/mL calculation
30 and 300
What is selective media?
Media that contains compounds that selectively enrich and/or selectively repress the growth of certain organisms while not affecting the growth of others
What is differential media?
Contains an indicator that differentiates the occurence of specific chemical reactions such that groups of bacteria can be differentiated from each other based on their ability to carryout that reaction
What is a fastidious organism?
An organism that is “delicate” and requires more specific conditions to grow
What are the selective and/or differential properties of MacConkey (MAC) agar? List the selective and differential ingredients, and the indicator in the plate
Also provide the source of macronutrients
PINK PLATE (neutral red indicator)
Selective: Selects for Gram- bacteria
- Selective ingredient: bile salts and crystal violet inhibits the growth of most G+ microbes
Differential: Lactose fermenters turn red, non-lactose fermenters become colourless/pale
- Differential ingredient: lactose
Source of macronutrients: Peptones and yeast extract
What are the selective and/or differential properties of SF agar? List the selective and differential ingredients, and the indicator in the plate
Also provide the source of macronutrients
PURPLE PLATE (Bromocresol purple indicator)
Selective: Inhibits most G- bacteria, selects for Enterococci
- Selective ingredient: Sodium azide, inhibits the electron transport chain in many Gram-negative bacteria
Differential: Enterococci present (ferment glucose)= yellow colonies with yellow-brown zones. Enterococci absent (non-fermenters) = medium remains purple
- Differential ingredient: dextrose
Source of macronutrients: Peptones (tryptone)
What are the selective and/or differential properties of Mannitol Salts (MSA) agar? List the selective and differential ingredients, and the indicator in the plate
Also provide the source of macronutrients
RED PLATE (Phenol Red indicator)
Selective: INHIBITS non-halophiles and G- (non-Staph)
- Selective ingredient: NaCl (high salt concentration), which most bacteria cannot tolerate, but Staphylococci can
Differential: Mannitol fermenters (e.g., Staphylococcus aureus) produce acid = medium turns yellow; Non-mannitol fermenters (e.g., Staphylococcus epidermidis) = no acid = medium stays pink/red
- Differential ingredient: Mannitol
Source of macronutrients: Peptones and beef extract
Describe the Ames test
There is a correlation between carcinogenesis and mutagenicity. Salmonella enterica that is auxotrophic for histidine and lacks DNA repair enzymes (to prevent the correction of DNA injury) is exposed to a chemical agent. After chemical exposure and incubation on histidine-deficient media, the rate of reversion to prototrophy is determined by counting the number of colonies that can grow (each colony is a His+ revertant) . This value is compared to a negative control (paper disc is placed in sterile water) and a positive control (paper disc is placed in a carcinogen like sodium azide)
Why are trace amounts of histidine present in the Ames test plates?
Allows His auxotrophs to grow (a little bit), to allow for mutations to actually occur in the first place to make them grow way more than the negative control (if the compound is carcinogenic)
How to calculate the number of induced mutations in an Ames test?
of colonies on plate - # of colonies on negative control
If bacteria grow on an incubated plate (at 37 degrees C) after initial exposure to 80 and 100 degrees C, what does this likely mean? Why would the same bacteria not grow after being incubated at 55 degrees C (not exposed to 80 and 100 degrees C)?
The bacteria formed endospores in 80 and 100 degrees C, and were able to grow upon incubation at 37 because 37 is favourable for them.
Wouldn’t be able to grow at 55 degrees because 55 is unfavourable for them (the organism isn’t a thermophile). There was no opportunity for the organism to form spores in the first place, but also you can’t tell if spores formed or not because there’s never a return to favourable conditions
What does an acid-fast stain identify? What is a positive result? A negative result?
Identifies if a cell can produce mycolic acid or not
Positive result = cells are red
Negative result = cells are blue or green
What are the 4 steps for an acid-fast stain?
- Prepare a bacterial smear
- Flood the smear with Kinyoun carbolfuchsin. Let the stain sit on the slide for 10 mins (without heat)
- Rinse the slide with water to remove excess stain. Remove excess water by tapping the slide gently on the edge of the staining dish.
- Decolourize with acid-alcohol until the run-off is clear. Rinse with water
Flood the smear (counterstain) with Brilliant Green. Let the stain sit on the slide for 1 min. Rinse with water, blot dry, and observe on 100x objective.
What are the 5 steps for an endospore stain?
- Prepare a bacterial smear
- Place the slide on a heating rack. Cover the smear (not the whole slide_ with a piece of paper towel and moisten the paper towel with Malachite Green. Steam for 10 mins
- Keep the paper moist with stain. The stain will evaporate throughout the procedure, so you will need to constantly add stain, such that the paper towel does not dry out and stick to the smear.
- Remove the paper towel with forceps and discard in the beaker. Use the water bottle to rinse the slide to remove excess stain.
- At your bench, counterstain with safranin for 1 minute. Rinse with water, blot dry, and observe under 100x objective
What is are the three cardinal temperatures?
The temperature range within which the growth of microorganisms is observed. This is unique to each species
1. Minimum temperature
2. Optimum temperature
3. Maximum temperature
What is the pH range of an organism defined as?
The difference between the maximum and minimum pH at which the organism will grow.
How do metals like copper sulfate inhibit microbial growth?
Ions can bind to proteins within the cell, damaging essential cellular processes and causing proteins to precipitate
What does it mean if there’s a greater zone of inhibition surrounding a paper disc with copper sulphate than the zone of inhibition surrounding a paper disc with silver nitrate?
The bacteria on the plate are more susceptible to the copper sulphate
How do you inoculate a thioglycolate medium?
Aseptically inoculate 200 µl into a thioglycoate broth tube below the upper pink layer
How do you inoculate a deep shake culture?
Aseptically inoculate 200 µl into a tube of molten tryptose agar. Mix gently by rotating the tube between the palms of your hands.
How is an oxidase test done? What do the results mean?
Scratch some bacteria on the end of an oxidase stick.
Cytochrome c oxidase is an enzyme within the aerobic electron transfer chain that reduces molecular oxygen with electrons from cytochrome c as the last step in the respiratory chain (electrons flow from cytochrome c to cytochrome c oxidase to oxygen)
TMPD is present on the oxidase stick, and it donates electrons to cytochrome c which is only oxidized if cytochrome c oxidase is active. Positive result = the oxidized TMPD will turn blue/purple (indicates that the bacteria is aerobic)
Negative result: If cytochrome c oxidase is not present, the TMPD will remain colourless
How do you do a catalase test and what do the results mean?
Transfer a mass of cells from a single, isolated colon of your unknown into two drops of H2O2 on a clean glass slide
Catalase converts H2O2 into water and O2 gas. Positive result= bubbles form, catalase present. Negative result = bubbles don’t form, catalase isn’t present.
Assign the following as loop inoculation or stab inoculation:
1. H&L test
2. Litmus milk
3. MR-VP test
- Stab
- Loop
- Loop
How do you do a nitrate reduction test and what do the results mean?
Loop inoculate a colony into the broth, be careful of the Durham vial.
Turbidity in the tube indicates organism growth, but only the
presence of a bubble in the Durham tube is a positive result
for denitrification by nitrate respiration (NO3- -> NO2- -> N2 gas eventually)
- Note: even if an organism can do nitrate reduction (using NO3- as a terminal electron acceptor) a bubble will only appear if it does full denitrification. Turbidity can appear for both positive and negative results.
Psychrophile temp. range
<15 degrees C
Mesophile temp. range
15-45 degrees C
Thermophile temp. range
45-80 degrees C
Hyperthermophile temp. range
> 80 degrees C
What is one molecular adaptation that allows organisms that grow in cold environments (less than 5 degrees C) to survive?
Psychrophiles have an increased abundance of unsaturated fatty acid talks in their inner cell membrane, which increases membrane fluidity at low temperatures as a result of kinks in the membrane
Neutrophile pH. range
5.5-7.9
Alkaliphile pH. range
> =8
Acidophile pH. range
<5.5
How do organisms that live in very high pH environments carry out membrane bioenergetic mechanisms such as flagellar rotation?
Alkaliphiles use sodium motive force to carry out membrane bioenergetics. This is established through an H+/Na+ antiporter, where H+ is pumped into the cell and Na+ is pumped out. Na+ can then travel down its concentration gradient (e.g. through ATP synthase or the Mot proteins for flagellar rotation) to power the membrane bioenergetics
What are 3 ways that E.coli gain heat resistance in food?
- Colanic acid: Polysaccharide that forms a mucoid matrix on cells
- SurA/PpiD: Reduces folding of outer mebrane proteins and induces a heat stress response
- Porin NmpC: A channel that contributes to “compatible solute” accumulation (it’s hydropilic, allows transport of hydrophilic solutes to regulate osmotic pressure under heat)
What is biofiltration? How does it work?
A biological wastewater treatment process that uses microorganisms to break down pollutants in sewage
1. Raw sewage comes into contact with media
2. Biofilm forms and contaminants attach
3. Pollutant breakdown
4. Pollutant transformation and removal
What are 4 challenges with biofiltration when battling biofilm in industry?
- Biofilms on microplastics (have a greater survival)
- Can compromise biosecurity of aquatic ecosystems
- Leads to the accumulation of sludge in soil environments
- Alters microbial ecology
What do 33% of deaths due to biofilms in food come from?
Listeria
What is the goal of the extreme microbiome project (XMP)?
To identify and characterize extremophiles from some of the harshest conditions on earth
What is a challenge of the XMP?
Extreme regions are hard to access, and it’s expensive to access them
Why do we care about thermophiles in the XMP?
Lead to the discovery of Taq polymerase, used in PCR. This discovery spurred the scientific community to keep investing in extremophile research
What are 3 useful products from fermentation in food production?
Lactic acid, acetic acid, propionic acid
What is the most common food fermentation? What organism carries out this fermentation?
Fermented dairy by Lactobacillus
What is first generation bioethanol as a next-generation fuel?
Fuel derived from high sugar/starch
What is second generation bioethanol as a next-generation fuel?
Derived from non-edible lignocellulose rich biomass (we don’t eat lignocellulose so this is readily available)
What are 4 harmful effects of oil spills?
- Decreases penetration of sunlight
- Decreased CO2 in water
- Contaminates food sources
- Causes reproductive issues
What is bioremediation?
The general process of using microbes, plants, or enzymes to remove or neutralize pollutants from a contaminated site (broad umbrella term)
What is bioaugmentation?
Adding specific strains of microorganisms to a contaminated site to enhance the cleanup process.
What is biostimulation?
Stimulating native microorganisms by adding nutrients (nitrogen, phosphorus) or oxygen to enhance their pollutant-degrading activity (e.g. Adding fertilizer to stimulate bacteria that break down petroleum)
Upon adding 3 drops of Methyl Red indicator to an MR-VP tube, what is a positive result and what is a negative result? What do these results mean?
Positive = red
Negative= remains yellow
Positive means that acids are present and indicates that mixed acid fermentation is carried out by the organism
How do you carry out a VP test? What is a positive/negative result and what do these results mean?
Vortex the tube well and add 15 drops of alpha-naphthol followed by 3 drops of KOH. Vortex the tube and let it sit for 10 minutes. Acetoin (a product of butaenediol fermentation_ oxidizes to diacetyl, which
reacts with peptones, producing a red pigment near the top of the tube (positive result)
Negative result = The medium turns a brown/copper colour
Positive result indicates the presence of butanediol fermentation
What do the deep shake results mean?
The location of turbidity (growth) that develops is an indication of the organism’s aerotolerance. If gas was produced in the anaerobic portion of the tube, then cracking of the agar may be observed (indicates that the organism carries out fermentation in the anaerobic portion)
What do the thioglycolate medium results mean?
Turbidity at top= aerobic
Turbidity at bottom = anaerobic
Equal turbidity = aerotolerant anaerobe
Some turbidity closer to top= microaerophile
Facultative anaerobe= More turbidity near the top
In an H&L glucose test, what does it mean if there’s yellow at the top of the unsealed tube but no yellow in the tube with mineral oil?
The organism is aerobic
In an H&L glucose test, what does it mean if there’s yellow throughout both the unsealed and sealed tubes?
The organism is a facultative anaerobe
In an H&L glucose test, what does it mean if there’s yellow at the bottom of the unsealed tube and throughout the entire sealed tube?
The organism is anaerobic
What does it mean if an H&L glucose tube turns blue?
The organism cannot utilize carbohydrates but can grow in the medium utilizing peptones (the organism produces alkaline end products)
- This is considered a negative result for carbohydrate utilization
What is the litmus milk test used to test for?
Evaluates an organism’s ability to metabolize lactose, casein (milk protein), and reduce litmus (a pH and redox indicator) in a milk-based medium.
What does a control litmus test medium look like?
Lavender purple
What does a light pink litmus test result indicate?
Acid production, indicates lactose fermentation with production of acidic end products
- LITMUS TURNS PINK
What does a half light pink (on top) and SOLID white (on the bottom) litmus test result indicate?
Acid + coagulation reaction (media acidification due to LACTOSE FERMENTATION on top precipitates solid casein)
- gas production can create fissures in the coagulated casein
- LITMUS IS REDUCED, INDICATED BY THE WHITE COLOUR
What does a blue litmus test result indicate?
Alkaline reaction, PARTIAL casein (milk protein) digestion with production of alkaline end products (ammonia)
- LITMUS TURNS BLUE
What does a layer with decreased opacity at the top and translucent yellow layer on the bottom litmus test result indicate?
Peptonisation (the medium LOSES ITS OPACITY at the top of the tube, and casein is digested leaving whey)
- basically, organism FULLY DIGESTS casein but uses peptones to grow in the media instead of lactose
Define cidal antimicrobial substances
Concerned with the killing of microorganisms
Describe static microbial substances
Growth-inhibiting
Define lytic antimicrobial substances
Cell lysing (SPECIFICALLY LYSING NOT JUST KILLING)
Difference between antiseptics and disinfectants?
- Antiseptics are cidal, static, or lytic against microorganisms but are still safe enough for use on living tissues
- Disinfectants are more potent cidal or lytic agents that destroy nearly all microorganisms but can be applied only to inanimate material because of their toxicity
What is the phenol coefficient and how do you calculate it? What does a coefficient >1 indicate? What about <1?
The ratio of a test agent’s effectiveness to phenol’s effectiveness.
Phenol coefficient = reciprocal effective dilution of test disinfectant/reciprocal effective dilution of phenol
The agents are tested against the same organism under the same conditions.
A coefficient >1 indicates that the test disinfectant is better than phenol
A coefficient <1 indicates that the test disinfectant is not as effective
What is an effective dilution?
The dilution of a test agent that completely inhibits growth at 10 mins exposure, but not at 5 mins exposure
In a Kirby Bauer Disc Diffusion test to test antibiotic effectiveness, what would a large zone of inhibition indicate? What about a small zone of inhibition? Also briefly explain how to prepare this test
Prepare a spread plate using a liquid culture of the organism of interest, then add Kirby Bauer Discs to each quadrant containing a different antibiotic.
Large zone of inhibition = microbe is susceptible to the antibiotic.
Small zone of inhibition = microbe is resistant to the antibiotic
In the transposon mutagenesis experiment, why was the plasmid in the donor E.coli S17-1 strain said to be a “suicide vector”?
The plasmid is unable to replicate in Rhizobium and will be lost as the Rhizobium replicates following conjugation. The transposon with resistance to gentamicin would integrate itself into the host Rhizobium though, allowing the recipient cells that have undergone transfer to be plated on a gentamicin plate.
What does a low MOI ensure?
Ensures near 100% adsorption, such that every virion attaches to a different host cell
Adsorption period on one-step growth curve
The virions attach to host cells and inject their DNA into the cytosol (attachment and penetration)
Eclipse period on one-step growth curve
When the phage replicates (synthesis)
- No virions are detected in the medium
Maturation period on one-step growth curve
When the progeny phage particles are assembled
Latent period on one-step growth curve
Eclipse and maturation periods together
Generation time on one-step growth curve
The elapsed time between the start of infection and the end of cell lysis
What is a colony forming unit (CFU)?
A measurement unit used in microbiology to estimate the number of viable microorganisms
- 1 CFU = 1 VIABLE cell or a cluster of cells capable of growing into a visible colony on an agar plate under the right conditions.
What is a plaque forming unit (PFU)?
A measurement used in virology to quantify the number of infectious virus particles in a sample.
- 1 PFU = 1 infectious virus particle capable of infecting a host cell, replicating, and creating a plaque (a clear zone where host cells have been destroyed).
Why did 0 E.coli S-17 colonies grow on the TY+Sm+Gn plate?
E. coli has a plasmid with the transposon that has antibiotic resistance to gentamicin (Gn), but is not resistant to streptamicin (Sm)
Why did 0 Rhizobium VF39 colonies grow on the TY+Sm+Gn plate?
Rhizobium has a chromosomal gene with antibiotic resistance to streptamicin (Sm), but does not naturally contain resistance to gentamicin (Gn)
Why did Rhizobium VF39 colonies grow on the TY+Sm plate
Because the Rhizobium has a streptamicin (Sm) resistance gene in its chromosome
How to calculate transposition frequency?
of potential hosts (i.e. # of cells on plate that allows for host growth, e.g. the Ty+ Sm plate for Rhizobia)/# of transposed cells (i.e. the number of cells, like Rhizobia, that grow on the TY+ Gn + Sm plate after exposure of donor cells to recipient cells)
What is the frequency of transposition defined as?
The number cells that underwent both conjugation and transposition per potential host (Rhizobium won’t get Gn resistance if transposon doesn’t integrate into Rhizobium chromosome, aka transposed since the plasmid with the Gn-resistant transposon is a suicide vector)
True or false: an organism can be positive for both MR and VP test at the same time
False; organism can’t carry out both mixed-acids and butanediol test
(Disinfectants/antiseptics) are generally more effective than (Disinfectants/antiseptics)
Disinfectants, antiseptics
(Gram-negative/Gram-positive) bacteria are more resistant to disinfectants
Gram-negative
(Gram-negative/Gram-positive) bacteria are more resistant to antibiotics, as seen from the Kirby-Bauer antimicrobial susceptibility test
Gram-positive
How to calculate eclipse phase of a single-step growth curve
End of adsorption (so end of decrease in titer) to the start of assembly (first time phages are detected again)
- use the premature lysis curve for this
How to calculate the latent period of a single-step growth curve
End of adsorption (taken from premature lysis curve) to the end of cell lysis (start of plateau, taken from natural lytic cycle)
What is the theoretical burst size and how do you calculate it?
The theoretical burst size is the size of the host genome divided by the size of the phage genome (i.e. how many bacteriophages you can build from the original host genome)
Theoretical burst size = size of host genome/size of phage genome
If the genome of E. coli is composed of 4600 kbp of dsDNA and the DNA genome of the T4 phage is 169 kb, what is the theoretical burst size?
4600 kbp/169 kbp = 27 phages can be made per cell (units just phages per cell
What is the actual burst size of a phage and how do you calculate it?
Burst size: the average number of phages released per cell
Burst size = # of released phages (taken from natural lysis)/# of original phages (taken from control)
Why does the actual burst size of a phage tend to be bigger than the theoretical burst size?
There are other sources of nucleotides outside of the chromosome
If the number of released phages is 141,200 from the natural lysis treatment and the number of original phages taken from the control treatment is 2150 (taken from the same time), what is the actual burst size?
Burst size = 141,200/2150 = 60
What is MOI and how do you calculate it at t=0?
MOI is the ratio of phage particles to bacterial host cells in a culture at a given time
MOI= # of phage particles (taken from control)/# of available bacterial cells
If the number of phage particles given from the control treatment at time 0 is 2200 and the number of available bacterial cells is 10^7, what is the MOI?
MOI= 2200/10^7 = 0.00022
How would you calculate the MOI at t=40?
Find the number of cells available at t=40 by subtracting 10^7-2200 (initial titer taken from the control)=
9 997 800
Then calculate MOI= titer at t40 from either natural lysis or premature lysis/# of available cells at t40
What information does the control provide in the single-step growth curve? (2)
- The original titer of the viral culture
- It also shows that the titer does not change without host cells present
How do you carry out a serial dilution?
900 µL of water in each tube. Aliquot 100 µL of stock broth into first tube. Take 100 µL of that tube and put it into the next tube. Keep going until the last tube. Make sure to vortex tube each time.
- Each transfer results in a ten fold decrease in concentration
What is a phage cocktail?
A solution containing a combination of multiple types of phages that can lyse the target bacteria. Allows treatment for multidrug-resistant (MDR) bacteria
What diseases have been shown to be effectively treated using phage cocktails?
UTIs, cystic fibrosis, life-threatening extensively drug-resistant Pseudomonas aeruginosa infection
What are some future steps for using phage cocktails to treat disease?
Cancer treatment, inflammation modulation, vaccine development
Where are used glass pipettes discarded?
the pipette bucket located on the side bench
Why is immersion oil required for the 100x objective lens but not the others?
Immersion oil reduces light refraction at 100x, improving resolution. Air gaps scatter light at high magnification.
