Flashcards in PA20014 applied microbiology flashcards 2 Deck (52):
You may do a sterility test, and conclude no growth is present, therefore product is sterile, however, you must remember that there are some bacteria that are impossible to grow, as we do not understand the conditions they require, therefore we may not pick up on them.
Remember nothing is 100% bacteria free!
The Sterilisation process may be effective for some, but ineffective for other microorganisms.
What does a bactericidal agent do?
Kills bacteria, so it will reduce the current number back down the the minimum. Bacteria are killed by these agents.
What does a bacteriostatic agent?
These just inhibit growth of bacteria (don't kill). They won't reduce the current number of bacteria, they just result in no further increase in number.
What sterilisation method should you use for anything heat sensitive?
Esp if you have a clear solution that's completely soluble
What is a good reference to use for validation of sterilisation processes?
Spores of bacillus stearothermophilus
The most resistant type of organism you can use is a bacterial endospore
If these have been killed, everything will have been killed
What are VBNCs?
Viable but not culturable microorganisms
Ie they are living but won't grow when put on media that normally supports growth.
These can produce endotoxins which can damage a drug and human.
Got to watch out for these!
Microbial inactivation follows ______ kinetics (ie killing microbes off)
First order kinetics
Surviving fraction curve decreases exponentially
What is the definition of the D value?
The TIME (or dose of radiation) required to reduce the number of viable cells by a factor of 10
The TIME (or dose of radiation) required to reduce the number of viable cells by 90%
The TIME (or dose of radiation) required to achieve a 1 log reduction in the number of viable cells
The higher the D value of a particular microorganism, the ______ the resistance.
As this means it takes longer to reduce the number of viable cells by a factor of 10
Less resistant organisms will have a _____ line as their survivor curves, as their D value is a _____ time.
Definition of Z value?
The temperature increase required to give a ten-fold reduction in the D value
The thermal destruction value
What graph do you plot to determine Z value?
Log of D value (y axis) against temperature ( x axis)
What is the usual range of Z values?
If you got an answer of say 20, you've probs gone wrong!!
D and Z values tell you about the resistance of a population but don't tell you about sterility
SAL and PNSU's are probabilitys of sterility.
They have to be expressed as a probability as there is no way we can tell if we have killed off every microorganism
Difference between SAL and PNSU?
SAL refers to the whole solution / whole batch, so it needs to be less than 1 microorganism per every 1 million (10^6) microorganisms
The PNSU refers to number of units in a batch. So this much be no more than 1 unit per 1 million units can be non sterile.
How do we calculate the D value of non linear curves?
The method changes with non linear curves
We work out the gradient of the straight part of the line using gradient=-k/ 2.303
We then use: D value = 1/ gradient To work out D value
How would you go about ensuring critical items, semi critical items aNd non critical were clean enough? I.e What method would you use for each?
Can't use disinfectants
You need to clean and sterilise using chemical sterilants and heat
Clean and use High level disinfectants
these are items that are going to come into contact with the patient, but not in contact with mucous membranes
Clean these and use a low to medium level disinfectant
Why is it important to clean a surface before disinfecting it?
This is because any Dirt, blood, pus, food (all organic matter)present on the surface can actually deactivate your disinfectant, so cleaning is important to make your disinfectant work!
What will require high concentrations of disinfectants and longer exposure times?
High bio burdens
BIOFILMS: these are really resistant, the extracellular matrix is difficult to penetrate by the disinfectant. We need high level disinfectants for biofilms an long exposure times
What are the most resistant type of bacteria?
Spores of bacillus stearothermophilus are also very resistant
Which are more resistant to disinfection: Gram positive or gram negative bacteria? Why?
Gram negative more resistant,
They have an outer membrane that is impermeable to outer chemicals, making it harder for a disinfectant to get in!
Apart from organic matter, what other thing can decrease activity of disinfectants?
Divalent cations such as Mg2+ and Ca2+.
These stabilise the cell wall of organisms, and block disinfectant absorption sites on the outer cell.
Things are added to disinfectants to remove these cations and stop them being a problem.
What properties do you need to think about when using a disinfectant, (hint; microbial killing is essentially a chemical reaction!)?
Dilution (some disinfectants are much more susceptible to being inactivated by dilution than others)
Exposure time (disinfectants will only work when they're wet,☂as water helps the disinfectant to absorb onto the cell surface and be taken into the cell) need to consider exposure time; don't want disinfectant to dry out)
What will a higher concentration of disinfectant increase and decrease?
Decreases exposure time
How do we work out dilution coefficient of a disinfectant ?
✎plot a graph of Log D value(y axis) against Log concentration of disinfectant.
✍ then work out gradient of the line, the gradient will be your dilution coefficient.
✎✍ remember gradient is change in Y axis so change in D values, over change in X axis so change in concentrations
How can we tell how effected a disinfectant is by dilution?
Calculate the dilution coefficient
The bigger the number, the more effected it is by dilution.
This is because D values must be quite large, ie it takes longer to kill 90% of organisms as the disinfectant isn't as good on dilution!
The higher the dilution coefficient, the larger the reduction in potency, the more effected the disinfectant is by the dilution!
What does a small dilution coefficient indicate?
The chemical isn't that effected by dilution
Dilution does not increase the D value
Dilution does not inactivate it or reduce its potency by much ☺
How does temperature effect disinfectants?
Increasing temperature will increase cidal activity of the disinfectant (it's killing ability) ☠☠☠
Increasing temp will increase killing Ability of different disinfectants by different amounts.
Be careful: if temp is too high your chemical may evaporate into a gas and you'll need an extractor fan!!☢☢
What is the infection control committee in hospitals?
Nurse, microbiologist, and a pharmacist, they draw up a policy staff must stick to to keep hospital contamination down.
When applying disinfectant, what is the double bucket technique?
You have a clean bucket and a dirty bucket , both have disinfectant in, use clean bucket to apply disinfectant and dirty bucket to squeeze your mop in.
What is the purpose of disinfectant rotation?
So bacteria don't build up a resistance to one alone, use 2 different types of disinfectant and rotate the use, less likely for resistance to develop.
How do you carry out a suspension test? testing how well a liquid disinfectant works
several different dilutions of disinfectant
Added to standardised bacterial suspension in water
Add albumin, source of protein which mimics organic, dirty conditions
Do at a set temperature
At a specific time , remove sample, neutralise disinfectant (stops it working!), determine the viable count!
Then calculate the concentration of disinfectant required to kill 99.99% bacteria
What do you need to perform Seperate tests for when doing suspension tests to test how well your disinfectant it working?
Seperate test for each species of bacteria
How do you carry out the European suspension test to see how well a liquid disinfectant is working?
Specific to bacteria
Do Seperate test for each bacteria species
Start with bovine serum albumin in water
Neutralise (stops disinfectant working)
Determine conc of disinfectant required to give a 5 log kill in 5 minutes at 20 degrees
What is minimum inhibitory concentration (MIC) test?
This is the lowest concentration of disinfectant (or antimicrobial agent) that shows no growth of bacteria.
So lowest concentration that doesn't show cloudiness.
Need separate MIC tests for different microorganisms.
You score for growth ✔✔✔✔✔, as soon as you get a non cloudy tube, this is your MIC
What do you need to remember to do with the MIC test and suspension test?
Do a different/ separate test for each species of microorganism!!!
What is the disc susceptibility test?
If your microorganism is sensitive to the disinfectant you get a zone of inhibiton around the edge where no growth has occurred. You can compare these zones of inhibition to see how sensitive your organism is to the disinfectant. If your test organism is sensitive to the disinfectant then you will get growth right up to the edge of the disc!
What are resident bacteria?
Bacteria permanently stuck to hands, and skin, live here.
What are transient bacteria?
Bacteria picked up from touching things etc
What tests do we use to evaluate antiseptics?
Time kill , determines viable count at time intervals (eg of 0-30 mins)
Can also do an MIC test (minimum inhibitory concentration)
Which tests are quantitive and which are qualitative for testing effectiveness of disinfectants and antiseptics?
Suspension tests and time kill are quantitive, as you're determining viable count, so the number of microorganisms present at the end.
MIC (minimum inhibitory concentration) is qualitative, as you're only determining either growth or no growth, you're not determining any viable counts
What should preservatives do for sterile products and non sterile products?
For sterile products they should KILL ALL microorganism that are introduced, hence make them sterile ☠☠
For non sterile products, they should just limit the growth of any microorganisms introduced, ie keep them below a certain level
In what kind of materials (packaging) can no specific absorption of preservatives occur therefore should try and avoid?
Rubbers and plastics
Should try to use glass where a preservative is used
What type of bacteria can contaminate all 3 types of product: eye products, oral products, topical products?
Pseudomonas aeruginosa and staphylococcus aureus
3 environmental factors that may determine how likely a product is to become contaminated?
Nutritional availability (if a carbon source is in your product; attracts bacteria)
Moisture content (microorganisms love water, but preservatives must work in aqueous phase)
Storage temp (storing at room temp makes contamination more likely, but your preservatives may not work well at lower temps, usually higher potency at higher temperatures)
What test do we use to evaluate preservatives?
The Challenge test
Assess activity of preservative when products in final container
Inoculate with 10^6 test microorganisms (Use bacteria for water based products like eye drops, and yeasts for syrupy products like cough medicine, as bacteria won't grow In these sugary conditions due to low moisture content but yeast will)
Incubate at specific temp for 28 days
Determine viable count
Check if in agreement with criteria for acceptance, expressed as the log reduction in viability
Do antiseptic hand washes get rid of both transient microorganisms and resident microorganisms?
No, only transient microorganisms.
You would have to use surgical hand scrubs or patient pre-op and skin care treatment to get rid of resident bacteria.
What is quorum sensing of bacteria?
Quorum= when their Is enough density of bacteria together for them to communicate
Quorum sensing= extracellular signals, called quorum sensing molecules, permit cell-cell communication, which induces initials matrix stages so bacteria start joining together, and therefore more chance of a biofilm forming. Bacterial cells communicate
How is disinfectants effectiveness against biofilms increased?
They can inhibit quorum sensing signals using inhibitors, bacterial cells can't communicate, therefore don't go into the initial matrix stages, and this stops them from growing in biofilms!☺
What is the role of mar A and mar R in E coli resistance?
Mar A is an activator of efflux pumps in the cell wall of E coli
Mar R is a repressor of efflux pumps in the cell wall of E coli.
Mutations in Mar R can occur, less repression of the efflux pumps, more activation, more antimicrobial pumped out of e coli, more resistance!
Bacteria may acquire genetic resistance, through chromosome mutation or gene transfer, what can this result in that contributes to increased resistance?
Length of O chain of LPS increase, more resistance.
Less and smaller PORINS: more resistance.
Increase in activity of efflux pump: more resistance, eg mutated Mar R gene in E coli resulting in increased activity of pump.