Lab Exam 2 Flashcards
Deep shake culture
The location of growth that develops is an indication of the organism’s aerotolerance.
- Cracks indicate growth in the anaerobic portion.
- Molten tryptose agar
Thyoglycolate medium
prepared by adding thioglycolic acid and glucose to semi-solid agar.
Oxygen gradient is created
- Resazurin = redox indicator
- Pink when oxidized, colourless when reduced.
Pink layer at the surface shows the depth to which oxygen has diffused into the medium.
The location of growth that develops is an indication of the organism’s aerotolerance.
Oxidase test
use the stick to pull off a small mass of cells from a single, isolated colony.
Cytochrome C oxidase is an enzyme in the aerobic etc = reduces molecular oxygen using the electrons from Cyt C.
reduced TMPD - colourless
blue/purple when TMPD oxidized
So blue/purple is observed if the reduced TMPD donates its electrons to oxidized cytochrome C (which only exists when cytochrome C oxidase is active.
Catalase test
Prepare a slide with two drops of hydrogen peroxide and then loop inoculate with the organism.
catalase functions by converting H2O2 to water and oxygen gas.
If organism has cells that produce catalase, oxygen will be seen bubbling in the water (positive result)
Four basic features of fermentation
Occurs in the absence of oxygen
energy producing electron transport is absent
is it much less energy efficient than respiration
metabolic intermediate
Homolactic fermentation
Production of lactic acid ONLY
These bacteria - first oxidize a carbohydrate to pyruvic acid via the EMP pathway (glycolysis). Pyruvic acid is then reduced almost entirely to lactic acid
acid production: litmus turns pink which indicates lactose fermentation with production of acidic end products
alkaline reaction
litmus milk turns blue
means that casein was partially digested with production of alkaline end products (ammonia)
litmus turns white
Indicates that reduction occurred
Litmus was reduced by a fermentation reductase enzymes
litmus milk medium solidifies
Media acidification precipitates casein.
gas production can create fissures in the coagulated casein
litmus milk peptonisation
the medium loses its opacity at the top of the tube
casein is digested leaving whey (translucent yellow colour)
multiplicity of infection
ratio of free phage particles to bacterial cells in a culture at a give time
low MOI ensures near 100% adsorption such that every virion attaches to a different host cell
MOI decreases to zero during penetration
eclipse period
When phage is replicating and synthesizing
is the time between the end of adsorption to the start of
assembly (i.e., last time phage detected to first time they are detected again on
premature lysis assay
maturation period
When the phage particles are assembled
MOI remains at zero here because the phage are still within the host cell
up until cell lysis
time from end of eclipse (when phage are first visible in the PRE-LYSIS curve) to the end of the spike segment in natural lysis
latent period
eclipse and the maturation periods together
from the start of infection to the beginning of lysis
end of adsorption and the end of cell
lysis (i.e., last time phage detected to start of plateau on natural lysis assay
generation time
is the elapsed time between the start of infection and the end of cell lysis
is the time between the start of infection and the end of cell
lysis (read from the natural lysis assay)
titer
known concentration of phage
burst size
represents the average number of new virions released from each infected cell
Methyl Red (MR) Test
- loop inoculate MR-VP broth tubes - incubate 24 hours - vortex tube and add 3-4 drops of Methyl Red
- low pH will give a red colour - indicating mixed acid production (positive result)
- higher pH will result in a yellow/orange colour. Indicates no mixed acid fermentation/minor acid production (negative result)
Voges Proskauer Test
loop inoculate MR-VP broth tubes - incubate 24 hours - vortex tube well –> add 15 drops of a-naphthol and 3 drops of potassium hydroxide - vortex and let tube sit for 10 mins
- If acetoin is present - it will slowly oxidize to diacetyl. The diacetyl then reacts with peptones, which produces a red pigment (positive result) –> red colour indicates butanediol production
- If acetoin is not present, the reagents will turn the medium a brown/copper colour (a negative result)
indicates no butanediol was formed
Anaerobic (nitrate) respiration
- respiration may occur with a final electron acceptor other than oxygen - results in the reduction of other compounds besides oxygens.
assimilitive nitrate reduction
When nitrate (NO3-) is reduced for use as a nutrient source (ie incorporating ammonia into amino acids and protein) - can occur aerobically or anaerobically
dissimilitive nitrate reduction:
restricted to bacteria and occurs when nitrate is used as an electron acceptor in energy metabolism (anaerobic respiration).
The ability to reduce nitrate to nitrite in the absence of oxygen is possess by many bacteria and the process is called nitrate reduction
denitrification
NO3- –> NO2- –> N2 (NO or N2) (anaerobic)
all gaseous products…
If nitrates are reduced: nitrogenous gases will form and become trapped at the top of teh Durham vial - generating a bubble (a positive result)
If nitrates in the medium are not reduced, nitrogenous gases will not be present and the Durham vial will not contain a bubble (a negative result). Media may still become turbid.
H and L glucose test
Tube has bromothymol blue pH indicator
glucose utilized - acid end products produced -acid results in a change in the the bromothymol blue indicator from green to yellow
If the organism can only ferment the carbohydrate, the colour change will only occur in the sealed (mineral
oil
If the organism can only respire the carbohydrate, the
colour change will occur in the unsealed media only. If the organism is facultative, then the colour change
will occur in both the sealed and unsealed media
If the organism cannot utilize the carbohydrate, but can grow in the medium utilizing peptones, the organism will produce alkaline end products. Excretion of these basic compounds into the medium results in the bromothymol blue changing from green to blue. This is still considered to be a negative result for carbohydrate utilization
Glucose/lactose tube turns blue - means
If the organism cannot utilize the carbohydrate, but can grow in the medium utilizing peptones, the organism will produce alkaline end products. Excretion of these basic compounds into the medium results in the bromothymol blue changing from green to blue. This is still considered to be a negative result for carbohydrate utilization
Transposons
a genetic element able to move from one site in a DNA molecule to another site on the same or a different DNA molecule
Carry the gene transposase that enables these jumping events
may carry genes that confer antibiotic resistance to the host organism
Frequency of transposition
tells us how many cells have been both conjugated and transposed
Number of bacterial cells in 1 ml of culture : total number of viable recipients (number that grew on the WT rhizobium plate (TY and SM)
DIVIDED BY NUMBEE OF CELLS /ML OF CULTURE to get a nice ratio
What is the theoretical burst size of the phage?
Size of the host genome divided by the size of the phage genome (ensure units are the same)
What information do you need to calculate the actual burst size?
Use the final PFU/ml / Initial PFU/ml (of the natural lysis)
This is larger than the theoretical burst size as the theoretical burst size does not take into account any extrachromosomal DNA that the virion may be packaging up
bubble indicates?
denitrification by nitrogen respiration
bacteriophage
A virus that infects bacteria
plaque forming unit
PFU. We count plaque forming units to estimate the titre of a particular solution containing phage (virion/ml)
it can be assumed that a plaque was cleared out by progeny of one original virion
viral titer units (PFU/ml)
What features can you obtain from a premature lysis curve?
eclipse period, maturation, latent period, and generation time.
What features can you
obtain from a natural lysis curve?
generation time, latent period, burst size
