asepsis 3

Question 1

what is bioburden?

Answer 1

•Most items come with a limited number of microbes already on them (bioburden)

Question 2

what are the categories in viable counting between alive and dead?

Answer 2

actively metabolising cell
cell with reduced metabolic activity
some metabolic activity and plasma membrane intact, but RNA content is reduced
plasma membrane intact,but no detectable metabolic activity
extensive damage to plasma membrane
cellular DNA degraded
cell fragments

Question 3

define sterile

Answer 3

sterile: An absolute term meaning free from all viable microorganisms–Not possible to have degrees of sterility

Question 4

define sterilisation

Answer 4

The process by which sterility is achieved, usually through the application of a biocidal agent or physical microbial removal

Question 5

define disinfection

Answer 5

The reduction of microorganisms on an inanimate surface

Question 6

what is a survivor curve?

Answer 6

Microorganism viability lost in exponential fashion following exposure to a killing process (not just a sterilization process)
•Independent of the starting number of organisms

Question 7

what do the 3 lines represent on a survivor curve?

Answer 7

•Continuous (A)•Initial reduced rate of kill during early stages (B)•Reduced rate of kill at low survivor levels (C)

Question 8

what microorganisms are the most resistant to sterilisation?

Answer 8

prions
spores
gram negative bacterial
small non-envoloped viruses
fungi
large non-envloped viruses
gram positive bacteria
lipid envloped viruses

Question 9

what are the 5 processes with standard methods for sterilisation?

Answer 9

steam( autoclave)
filtration
dry heat ( oven)
gas sterilisation
ionising radiation

Question 10

what happens in practical sterilisation?

Answer 10

procedures are designed and validated with “Overkill” in mind
•Start with a measurable amount and extrapolate

Question 11

in most cases what is the initial bioburden?

Answer 11

lower than the required level of kill needed (i.e. a 6 log reduction)

Question 12

what is the D value?

Answer 12

The decimal reduction time (D-value) originated from assessing the thermal resistance of microbes
•The D-value is the amount of time required to reduce the number of viable organisms by 10% (a 1 log reduction) at defined experimental conditions
•Only true in steam sterilization

Question 13

how is the D value usually quoted?

Answer 13

•Usually quoted with reference to the temperature tested•i.eD121of 5 minutes

Question 14

when do D values increase/decrease?

Answer 14

• D-value increases at lower temperatures

* D-Value decreases at higher temperatures

Question 15

how can D values also be quoted?

Answer 15

• D-values can be quoted for other sterilisation methods as well
• In these cases it may not be based on exposure time

Question 16

what is a z value?

Answer 16

change in temperature that produces a tenfold (1 log reduction) change in D value
For named microorganisms a Z value is a measure of its resistance to heat

Question 17

what do physical and chemical indicators show?

Answer 17

they do not guarantee sterilization only show procedural error/ equipment malfunctions

Question 18

what is a physical indicator of a preformance verification test?

Answer 18

• Measurement of physical parameters with mechanical or electronic recorders
• Includes things like temperature sensors, pressure sensors
• Will provide some form of log for quality documentation

Question 19

how many different classes of chemical indicators are there for steam sterilisation?

Answer 19

there are 6 different classes

Question 20

what do the different classes in steam sterilisation show?

Answer 20

class 1 only shows exposure to process- e.g. autoclave tape
class 5 is designed to react with all essential variables during a cycle e.g. thermalog
colour change is directly related to death curve of B

Question 21

What is the only type of indicator that shows that a process has killed organisms?

Answer 21

biological process

this is uaully for high resistance organisms

Question 22

what are the two different techniques used for sterility testing?

Answer 22

• Direct Inoculation

* Membrane filtration

Question 23

what media is used for sterility testing?

Answer 23

Soya-bean casein digest medium (or as you know it TSB)

•Allows for the growth of anaerobic and aerobic organisms

Question 24

when there is no growth over a 14 day period the product is sterile unless:

Answer 24

• Environmental monitoring of facility shows a fault
• There is a fault in the testing procedure
• There is growth in the negative control

Question 25

what is direct inoculation?

Answer 25

• As it sounds: Add product directly to media and incubate

* Preferred methods for materials that cant be easily filtered

Question 26

how does membrane filtration work?

Answer 26

• Lets large volumes of sample be filtered•Also can rinse membrane to remove inhibitory substances
• Helps to minimize human intervention

Question 27

how do you inturpurate sterility testing results?

Answer 27

•When looking at the sterility testing of a product the number of microbes don’t matter!
–If anything grows = fail
–If stays clear= pass

Question 28

what are pyrogens?

Answer 28

•Pyrogens are fever inducing substances;
–Endogenous pyrogens are usually cytokines which induce a shift in temperature regulation once they have exceeded a threshold
–Exogenous pyrogen are usually microbial in origin–For pharmaceuticals usually means endotoxins (LPS) from G-bacteria

Question 29

where do the different types pyrogens live?

Answer 29

•Both live and dead bacteria have the same pyrogenic potential

Question 30

what is depyrogenation?

Answer 30

•Depyrogenation is the process by which pyrogens can be removed or destroyed

Question 31

how cab pyrogens be removed?

Answer 31

•Pyrogens can be removed by;–Ion exchange chromatography–Ultrafiltration–Distillation

Question 32

how can pyrogens be destroyed?

Answer 32

•Pyrogens can be destroyed by;–Acid base hydrolysis–Oxidation–Heating–Sodium hydroxide

Question 33

what are the 3 main tests used to detect endotoxins and pyrogens?

Answer 33

• Rabbit pyrogen test (introduced to USP in 1942)
• Limulus amebocyte assay (introduced to USP in 1980)
• Monocyte activation test (introduced to EP in 2010)

Question 34

how does the rabbit pyrogen test work?

Answer 34

• Product initially injected into marginal ear vein of 3 rabbits (≤10ml/Kg)
• Number increases if product fails•Temperature monitored every 30 mins for 3h

Question 35

what are the limitations of rabbit pyrogen test?

Answer 35

• Not very sensitive
• Not very sensitive to some bacterial endotoxins (e.gLegionella)
• Variation in rabbit species response when standard endotoxin solutions used
• Incompatible with certain types of drugs (e.g. radiopharmaceuticals)

Question 36

what is LAL assays specific for?

Answer 36

• Specific for bacterial endotoxins
• 3 different types of LAL assay recognised;
• Gel clot technique
• Turbidimetric technique
• Chromogenic technique

Question 37

what does LAL assay rely on?

Answer 37

•Relies on the clotting cascade of horseshoe crab blood

Question 38

what is the problem with LAL assay?

Answer 38

factor C needs to be activated in order to generate the clot

other things can also be activated thus giving a false positive result

Question 39

how does monocyte activation test work?

Answer 39

Uses monocytes from human blood to detect pyrogens–Either cell lines, whole blood or peripheral blood mononuclear cells–Can be used as an alternative to Rabbits
•Relies on the monocyte to generate pro-inflammatory cytokines which can be detected by ELISA

Question 40

what are endotoxin limits? and how do you calculate it?

Answer 40

are based on the dose of the product given per hour

EL=K/M

K = minimum pyrogenic dose (i.e. the maximum IU of endotoxin which a patient can get without a toxic reaction)•M = maximum dose of the drug substance per person (or Kg) per hour