PA20014 applied microbiology Flashcards
Who devised a series of tests to assess germ theory of disease and mortality after surgery?
Robert Koch
Who discovered that growth of microbes was NOT through spontaneous generation, in 1860?
Louis Pasteur
Which two scientists were the drivers of how microbiology was to be done?
Louis Pasteur, Robert koch
What’s the problem with multi-dose containers?
They are used several times for different patients, should use single dose instead or use multi dose that have been sterilised by terminal autoclaving (sterilising the final container)
What was the sterilisation of topical and oral preparations triggered from?
Thyroid tablets found to be contaminated with bacteria.
Definition of sterile?
Free from all viable forms of life
It is expressed as a probability
What does the ‘negative state is hard to proove’ mean?
It’s harder to prove that there are no forms of life, it’s a lot easier to prove that there ARE forms of life present!
With the definition of sterile being free from all forms of life, one hitch is that we may not pick up all forms, how come?
When we do quality control, there is only a LIMITED AMOUNT OF MEDIA we can use to test for bacteria and this may not be sensitive to all forms of life,
Also some bacteria are impossible to grow and develop tests for as we don’t understand the conditions they require.
This is why we can’t say things are absolutely sterile; impossible to tell if we actually killed all forms of life.
What does ‘ the sterilisation process is not an all or nothing phenomenon’ imply?
Sterilisation will process like a first order chemical reaction, ie the population exposed to the sterilisation process will decrease in number EXPONENTIALLY, but the number will NEVER REACH ZERO.
This basically means you never kill all the bacteria all at once, and some will always survive.
Why don’t antimicrobial products state “this is 100% effective!”?
Number of bacteria left after sterilisation never reaches zero. You can’t prove that all bacteria have been killed off as you can’t detect some bacteria. They just kill most of the bacteria eg 99%.
What goes on the y axis of a fraction surviving curve? What does this mean?
N/ No
This is a percentage, N= number of bacteria still surviving, No= number of bacteria you originally started with.
It is the fraction of surviving bacteria/ microorganisms
What does bioburden mean?
The number of bacteria present in the product before sterilisation
If you want a better level of PNSU, should you sterilise for a longer or shorter time?
Longer time
Gets the probability of bacteria being present per unit right down.
What are the problems with long sterilisation times?
Drugs may start to degrade (due to high temps used)
The process is time consuming and expensive
What is the minimum required standard value of PNSU for most pharmaceutical products?
1 in 10^6
What type of bacteria can form spores?
Only SOME types of GRAM POSITIVE bacteria
What types of microorganisms are most likely to be contaminants ?
Bacteria and Fungi
What types of bacteria can be resistant to sterilisation?
Some types of gram positive bacteria that can form spores.
In what area of manufacture therapeutics are bacteria and fungi used in?
Recombinant DNA technology
What are the most stable/ resistant type of microorganism?
Prions.
What are the least resistant type of microorganism?
Multicellular organisms, then vegetative bacteria and fungi
The killing of _____ can be used to validate various sterilisation methods?
Bacterial endospores. This is because they’re very resistant to things like chemicals and heat therefore if you can kill the spores in your process then you can be sure other organisms are killed too.
What are prions?
Infectious agents composed of protein in a misfolded form.
Are all bacteria pathogenic?
No, most bacteria and non- pathogenic in fact!
But there are some pathogenic bacteria..
Which type of bacteria have a THICK peptidoglycan layer?
Gram positive
How many membranes do gram positive bacteria have?
ONE cytoplasmic membrane
What type of acids does the peptidoglycan layer in gram positive bacteria contain?
Lipoteichoic (longer ones) and teichoic acids
Which type of bacteria has a thin peptidoglycan layer? What is the intermembrane space called that this is present in?
Gram negative bacteria
The periplasm
How many membranes do gram negative bacteria have?
2, and outer membrane which contains PORINS and LPS, and and cytoplasmic membrane.
What is LPS? Where is it found & on which bacteria type?
Lipopolysaccharide is an ENDOTOXIN (/pyrogen) to which the immune system responds to.
It is found on the outer membrane of gram negative bacteria (sticks up and out of it)
What is vegetative growth?
When nutrients are available to bacteria they will happily grow and divide, this is vegetative growth
What triggers bacteria to form spores?
When nutrients run out, some gram positive bacterias survival strategy is to form spores.
What does the core of the prespore contain in maturation of a spore?
Contains DNA of bacteria.
What is the cortex in spore formation?
A thick layer of peptidoglycan between two membranes (outer and inner membrane of spore. Outer membrane obtained from mother cell by engulfment)
What is a mature bacterial spore called?
An endospore
What does spore germination entail?
If conditions are right, i.e. right amounts of nutrients and water are available, then the spore will germinate and form a normal bacterial cell once again. It is the process of a spore becoming a normal bacterial cell when conditions allow it too. Remember it forms a spore in the first place because conditions weren’t right.
How does a bacterial cell release the mature endospore ?
By cell lysis.
In which phase of bacterial growth does sporulation usually occur?
The stationary phase, this is the phase when nutrients have started to run out and bacterial cell division and growth has started to slow down. Spores form as a survival mechanism.
What happens during the exponential growth phase of bacterial growth?
Bacterial Cells divide spontaneously and rapidly as there is no shortage of nutrients in this phase.
What is a biofilm?
A layer/ community of cells that attach to a surface, that are much more resistant to things like antibiotics and biocides.
What happens once all the bacteria have grown inside the biofilm?
Dispersal. The bacterial cells are released/ dispersed from the biofilm so that they can go and take action.
How does the extracellular material of the biofilm contribute to bacterial resistance?
Bacterial cells are embedded in this extracellular matrix.
Extracellular film limits the penetration of antibiotics.
Biofilms are more difficult to phagocytose.
What are the five stages of biofilm formation?
Initial attachment to surface Irreversible attachment Micro-colony formation Maturation Dispersal
What’s a common medical device that biofilms grow in?
Catheters
Biofilm formation in industrial processes is know as ______?
Biofouling
What are the three types of fungi?
Yeasts
Multicellular Filamentous MOULDS
Macroscopic Filamentous fungi
What is the main method of reproduction of yeasts?
By budding/ fission
But they can form spores
What is the main method of reproduction of moulds?
By forming SPORES
They grow as masses of overlapping/ interlinking hyphal filaments
What are the two groups of viruses? What do they differ in structure by?
1) naked viruses, these are a protein capsid filled with nucleic acid
2) enveloped viruses; these have an additional membrane around the capsid. This membrane comes from the host!
Viruses are obligate intracellular. What does this mean?
They can only reproduce within a host, only inside host cells.
This is why they’re less of a problem with contamination, as they need a host to survive.
Protozoa don’t have cell walls. This means they don’t survive ___ well. This shows they are ____ stable than bacteria.
Drying
Less stable
Infectious prions are misfolded forms of protein. What is the code for this form?
PrPsc.
These forms can convert normal prions (PrPc) into the misfolded kind, forming aggregates which are insoluble and cause disease affecting the brain.
What is the disease caused by prions?
CJD ( creutzfeldt- Jakob Disease)
& Kuru ( related to CJD)
What is the most common form of contamination?
Non viable contamination.
Foreign material is the more common form of non viable contamination
What does non viable contamination mean and what are some examples?
Non living contaminants Examples are Particulates Toxins Metabolites Pyrogens (egLPS)
What are pyrogens?
any substance or agent that tends to cause a rise in body temperature and induce fever, such as some bacterial toxins.
Toxins/endotoxins can be pyrogens, such as LPS
Examples of viable contaminants?
Bacteria, fungi, viruses
What type of vegetative cells (bacteria) AREN’T sensitive to radiation?
Deinococcus radiodurians
Remember radio; radiation
Of a dinosaur with a c**k listening to Duran duran on the radio!!
Which are more resistant to sterilisation, large viruses or small viruses?
Small viruses are MORE resistant
Which are more resistant to sterilisation,fungal spore or bacterial endospores?
Bacterial endospores are more resistant
Very resistant to things like chemicals and heat
These spores are often used to validate sterilisation processes
Which are more resistant to sterilisation, prions or bacterial endospores?
Prions
These are the most resistant
Which are more resistant to sterilisation, multicellular organisms or bacteria?
Bacteria (single cellular) are more resistant
Multi cellular organisms are the least resistant things to sterilisation
Do all gram positive bacteria form spores?
No, only some not all
In a biofilm, cells are in close proximity. What does this allow?
Molecular signals can be exchanged between the cells that regulate behaviour
How do yeasts ( type of fungi) reproduce ?
Mostly reproduce by budding/ fission
But They can form spores!
How do moulds (type of fungi) reproduce?
Reproduce by forming spores.
They grow as masses of overlapping/ interlinking hyphal filaments
Are fungi eukaryotes or prokaryotes? What about bacteria?
Fungi; eukaryotes
Bacteria: prokaryotes
Which are more abundant, bacteria or viruses?
Viruses, these are very abundant
But remember they’re not much of a problem with contamination as they need a host to survive
What type of products would viruses contaminate?
Blood related products, tissue fluid products, as they can only survive in living cells.
For example HIV virus infecting blood products
What is spoilage?
Products damaged by presence of microbes. Breakdown of actives/ excipients etc can occur in products.
What products must be STERILE?
INJECTABLES and opthalmic preparations (eye preps)
What are the viable count limits for oral preps and topical preps? (for non sterile products)
Oral preps:
Must have less than 10^3 aerobic bacteria
Less than 10^2 fungi
Zero E.coli!!!
Topical preps:
Less than 10^2 bacteria
Less than 10^2 fungi
What are microorganisms that cause air contamination quite resistant too?
Drying
__% of the microbes contaminating water are gram _____
P______ contaminates fresh water
E_____&P____ contaminate sewage
98% , gram negative
Pseudomonas contaminate fresh water
E.coli and proteus contaminate sewage
3 types of water that are non sterile?
Potable mains water (tap)
Softened water (water with Mg and Ca removed)
De- ionised water (filtered/ treated to remove ions only)
What are the two types of sterile water, which one is used more for injections?
Distilled water (boiled then condensed to collect the pure water, ions and minerals all left behind) Reverse osmosis water (pyrogen free) it is used as water for injections
How can you minimise contamination of water in storage vessels and distribution systems?
Maintain water at 80 degrees
Circulate the water at a positive pressure (so it doesn’t become stagnant)
Decrease length of pipeline
Include a sterilisation system
What is the need of a Pre filter?
Protects the actual filter,
It has larger pore sizes than the actual filter, it will filter the bigger particles out so that they wont damage the actual filter with smaller pore sizes or push through any small particles. Actual filter will have much smaller pore sizes.
Raw material of a natural origin have high levels of microflora
Raw materials of a synthetic origin have low levels of microflora
Why would you add sugar/ syrup to a liquid or semi solid pharmaceutical being stored?
Sugars Reduce water availability (Aw)
Bacteria need water and moisture to grow ideally, reduce as much water that’s present as you can, store in dry conditions
What types of bacteria can buildings get contaminated by?
Aspergillus, penicillium, bacillus, clostridium
Remember clean rooms are CONTROLLED environments!!
Controlled temp, humidity, air flow, pressure, filtered air, constant monitoring, particle counts
