PA20014 applied microbiology flashcards 2

Question 0

What does a bactericidal agent do?

Answer 0

Kills bacteria, so it will reduce the current number back down the the minimum. Bacteria are killed by these agents.

Question 1

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.

Answer 1

Remember nothing is 100% bacteria free!

The Sterilisation process may be effective for some, but ineffective for other microorganisms.

Question 2

What does a bacteriostatic agent?

Answer 2

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.

Question 3

What sterilisation method should you use for anything heat sensitive?

Answer 3

Filtration

Esp if you have a clear solution that’s completely soluble

Question 4

What is a good reference to use for validation of sterilisation processes?

Answer 4

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

Question 5

What are VBNCs?

Answer 5

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!

Question 6

Microbial inactivation follows ______ kinetics (ie killing microbes off)

Answer 6

First order kinetics

Surviving fraction curve decreases exponentially

Question 7

What is the definition of the D value?

Answer 7

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

Question 8

The higher the D value of a particular microorganism, the ______ the resistance.

Answer 8

Greater

As this means it takes longer to reduce the number of viable cells by a factor of 10

Question 9

Less resistant organisms will have a _____ line as their survivor curves, as their D value is a _____ time.

Answer 9

Steeper line

Shorter time

Question 10

Definition of Z value?

Answer 10

The temperature increase required to give a ten-fold reduction in the D value
The thermal destruction value

Question 11

What graph do you plot to determine Z value?

Answer 11

Log of D value (y axis) against temperature ( x axis)

Question 12

What is the usual range of Z values?

Answer 12

9-12 degrees

If you got an answer of say 20, you’ve probs gone wrong!!

Question 13

D and Z values tell you about the resistance of a population but don’t tell you about sterility

Answer 13

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

Question 14

Difference between SAL and PNSU?

Answer 14

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.

Question 15

How do we calculate the D value of non linear curves?

Answer 15

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

Question 16

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?

Answer 16

Critical:
Can’t use disinfectants
You need to clean and sterilise using chemical sterilants and heat
Semi critical:
Clean and use High level disinfectants
Non critical:
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

Question 17

Why is it important to clean a surface before disinfecting it?

Answer 17

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! 

Question 18

What will require high concentrations of disinfectants and longer exposure times?

Answer 18

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

Question 19

What are the most resistant type of bacteria?

Answer 19

Mycobacteria

Spores of bacillus stearothermophilus are also very resistant

Question 20

Which are more resistant to disinfection: Gram positive or gram negative bacteria? Why?

Answer 20

Gram negative more resistant,

They have an outer membrane that is impermeable to outer chemicals, making it harder for a disinfectant to get in!

Question 21

Apart from organic matter, what other thing can decrease activity of disinfectants?

Answer 21

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.

Question 22

What properties do you need to think about when using a disinfectant, (hint; microbial killing is essentially a chemical reaction!)?

Answer 22

Concentration
Dilution (some disinfectants are much more susceptible to being inactivated by dilution than others)
Temperature
pH
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)

Question 23

What will a higher concentration of disinfectant increase and decrease?

Answer 23

Increases efficacy

Decreases exposure time

Question 24

How do we work out dilution coefficient of a disinfectant ?

Answer 24

✎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

Question 25

How can we tell how effected a disinfectant is by dilution?

Answer 25

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! 

Question 26

What does a small dilution coefficient indicate?

Answer 26

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 ☺

Question 27

How does temperature effect disinfectants?

Answer 27

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!!☢☢

Question 28

What is the infection control committee in hospitals? 

Answer 28

Nurse, microbiologist, and a pharmacist, they draw up a policy staff must stick to to keep hospital contamination down.

Question 29

When applying disinfectant, what is the double bucket technique?

Answer 29

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.

Question 30

What is the purpose of disinfectant rotation?

Answer 30

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.

Question 31

How do you carry out a suspension test? testing how well a liquid disinfectant works

Answer 31

 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

Question 32

What do you need to perform Seperate tests for when doing suspension tests to test how well your disinfectant it working?

Answer 32

Seperate test for each species of bacteria

Question 33

How do you carry out the European suspension test to see how well a liquid disinfectant is working?

Answer 33

Specific to bacteria 
Do Seperate test for each bacteria species
Start with bovine serum albumin in water
Add bacteria
Add disinfectant
Neutralise (stops disinfectant working) 
Culture plates
Determine conc of disinfectant required to give a 5 log kill in 5 minutes at 20 degrees

Question 34

What is minimum inhibitory concentration (MIC) test?

Answer 34

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

Question 35

What do you need to remember to do with the MIC test and suspension test?

Answer 35

Do a different/ separate test for each species of microorganism!!!

Question 36

What is the disc susceptibility test?

Answer 36

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!

Question 37

What are resident bacteria?

Answer 37

Bacteria permanently stuck to hands, and skin, live here.

Question 38

What are transient bacteria?

Answer 38

Bacteria picked up from touching things etc 

Question 40

What tests do we use to evaluate antiseptics?

Answer 40

Time kill , determines viable count at time intervals (eg of 0-30 mins)
Can also do an MIC test (minimum inhibitory concentration)

Question 41

Which tests are quantitive and which are qualitative for testing effectiveness of disinfectants and antiseptics?

Answer 41

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

Question 42

What should preservatives do for sterile products and non sterile products?

Answer 42

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

Question 43

In what kind of materials (packaging) can no specific absorption of preservatives occur therefore should try and avoid?

Answer 43

Rubbers and plastics

Should try to use glass where a preservative is used 

Question 44

What type of bacteria can contaminate all 3 types of product: eye products, oral products, topical products?

Answer 44

Pseudomonas aeruginosa and staphylococcus aureus

Question 45

3 environmental factors that may determine how likely a product is to become contaminated?

Answer 45

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)

Question 46

What test do we use to evaluate preservatives?

Answer 46

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

Question 48

Do antiseptic hand washes get rid of both transient microorganisms and resident microorganisms?

Answer 48

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.

Question 49

What is quorum sensing of bacteria?

Answer 49

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 

Question 50

How is disinfectants effectiveness against biofilms increased?

Answer 50

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!☺

Question 51

What is the role of mar A and mar R in E coli resistance?

Answer 51

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!

Question 52

Bacteria may acquire genetic resistance, through chromosome mutation or gene transfer, what can this result in that contributes to increased resistance?

Answer 52

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.

Question 53

What sterilisation methods can Z value be used for?

Answer 53

Only things involved heat, as it is a temperature , so only moist heat sterilisation and dry heat sterilisation.