Lecture 4: physical control methods Flashcards
terms used for assessing the efficacy of heat treatments
- thermal death point
- thermal death time
- decimal reduction time
- death rate constant
- z value
thermal death point
the lowest temp required to kill all cells in a standard suspension of bacteria in a liquid culture within a period of 10 mins
why do we need a control sector
to check the viability of experiment
thermal death time
the length of time required to kill all of the cells in a standard suspension of bacteria in a liquid culture at a given temp
decimal reduction time (D value)
the length of time taken to obtain a ten fold reduction in the number of bacteria in a standard suspension of bacteria in a liquid culture
equivalents of ten fold reduction
90% killed
1 log difference
what does K stand for
- the death rate constant, which is also the slope of the curve
K equation
K = 2.3/t x log10 (Nt/No)
t= time
Nt = number of surviving cells at time t
No = number of cells at zero time
z value
the temp required for one log10 reduction in the D value
filtration (mechanical removal)
- physical sterilisation method
- through filter with pores to small for microorganisms, but large enough to allow the liquid or air to pass through
- not killing them, putting a barrier in place
what is the average size of a pore
0.20 um to filter out bacteria and bacterial endospores
three different types of filters
- depth filter
- membrane filter
- nucleopore filter
depth filter
Structure: random array of overlapping fibres eg: paper or glass
thick = 5-7mm high
Advantage: high dirt handling capacity = can randomly trap a lot of cells in this structure
Disadvantage: bacteria get trapped in this structure randomly, bc its so thick it will retain some of the liquid we are trying to filter through it
How it would be used:
can be used in tandem, could put solution through this depth filter, hepa filter used to decontaminate the air going in and out of the safety cabinet
membrane filter
structure: a lot less random, has a definite pore size, more common type of filter in use, thin disc, like a sheet of paper, cellulose acetate or cellulose nitrate, acts like a sift, cells get trapped on the surface
advantages: definite pore size, thin so won’t retain any fluid
disadvantages: could get blocked (can be overcome by cleaning it regularly) or using it in tandem
how it would be used: tissue culture media
nucleopore filter
structure: definite pore size but not many pores, made of polycarbonate = super thin, rigorously engineered pore size
advantages -
disadvantages - very slow, low flow rate
how it would be used - preparation of samples for electron microscopy work
HEPA filters
- class II biological safety cabinets (protecting specimen, user, and environment from contamination)
- removes greater than 99.97% of particles that are 0.3um or larger, by physical retention and electrostatic interactions
examples of solutions that can be filtered
- tissue culture media
- serum
- antibiotic solutions
- gases
- filtered beer
types of radiation
- non ionising radiation
- ionising radiation
non ionising radiation
- ultra violet radiation
- wavelength of 260nm damages the DNA in the cell by forming pyrimidine dimers or direct protein damage
- applications include the sterilisation of benches and air
- not used to sterilise large volumes of liquids as it cannot penetrate these to a significnt depth
- does not kill bacterial endospores
bacteria repair systems
- nucleotide excision repair
- direct repair
- recombinational repair
- SOS repair
nucleotide excision repair
the UvrABC endonuclease enzyme removes damaged nucleotides. the resulting single stranded gap is filled by DNA polymerase I, and DNA ligase joins the fragments
direct repair
uses photoreactivation, visible light and photolyase (enzyme
- another way of repairing UV damage
recombinational repair
corrects damaged DNA using Rec A
SOS repair
a transcriptional repressor protein (LexA) is destroyed. Many genes involved. error prone
ionising radiation (x-rays, gamma rays)
- kills indirectly by inducing reactive chemical radicals (free radicals) by breaking individual molecules into ions, hence ionising radiation
- kills bacterial endospores
- involves very short wavelengths, therefore high energy –> causes atoms to ionise
what happens when y rays interact with water
- free radicals are formed
- hydrogen free radicals are strong reducing agents
- hydroxyl free radicals are strong oxidizing agents
ionising radiation advantages and disadvantages
advantages- ionising radiation is penetrating and therefore can be used to sterilise products even after they have been packaged
disadvantages - expensive to operate, requires elaborate safety precautions (lead shielding of operators)
