working with microbes

Question 1

What does aseptic technique mean?

Answer 1

Without microorganisms

Question 2

Why use aseptic techniques?

Answer 2

• Prevention of contamination of the laboratory by the organisms being handled
• prevention of contamination of the work with organisms from the environment

Question 3

What does aseptic techniques do?

Answer 3

• remove or kill microorganisms from hands, equipment and objects
• employ sterile instruments and other items
• remove risk of contamination of experiments, enviro and people by microorganisms

Question 4

what is autoclaving

Answer 4

pressurised steam steriliser

-autoclave conditions-121 degrees, 15psi, 15 mins

Question 5

what is autoclaving used to sterilise

Answer 5

-bacteriological culture media
•Glassware
•Equipment e.g. pipette tips

Question 6

name 3 physical ways of sterilisation

Answer 6

• heat sterilisation (dry or moist)
• filtration (asbestoses, membrane, sintered glass)
• radiation sterilisation (ionising or non ionising)

Question 7

dry and moist sterilisation in heat sterilisation

Answer 7

• moist heat sterilisation e.g. autoclave, boiling water, water heating, pasteurisation, pressurised steam
• dry heat sterilisation e.g. dry oven, incineration

Question 8

ionising and non ionising in radiation sterilisation

Answer 8

-ionising- x rays, U.V rays, cathode

non ionising- ultra violet

Question 9

name two chemical ways of sterililsation

Answer 9

gas and liquid

Question 10

name 2 gas ways of sterilisation

Answer 10

• formaldehyde

- ethylene oxide

Question 11

Name 4 liquid ways of sterilisation

Answer 11

alcohol
Halogens
Phenols
Aldehydes

Question 12

How do we know that what we have autoclaved is sterile

Answer 12

1) autoclave tape has strips which change colour when exposed to 121 degrees
2) the change in colour is permanent so in the lab you will know which equipment is sterilised
3) indicator valves also can be used to assess successful autoclaving

Question 13

What is pasteurization

Answer 13

A carefully controlled heating to reduce microbial load and it is associated with milk
It is developed to kill pathogenic microorganisms but it’s not sterilisation coz it doesnf kill all organisms.
The milk is passed through a head exchanger at 71c for 15s and cooled rapidly

Question 14

Types of radiation

Answer 14

Microwaves, ultraviolet, xrays, gamma Ray’s and electrons as well as heat is a form of energy

Question 15

ionizing radiation used for which pre packed plastic equip

Answer 15

Petri dishes
Spreaders
Syringes
(Use one and dispose)

Question 16

What is ionizing radiation

Answer 16

Energy to produce ions from other molecules

It can generate hydroxyl group (OH•) and hydride(H•)

Question 17

What does ionizing radiation do

Answer 17

It causes damage to the DNA and other macro molecules-call death
And uses electron beams, x-ray machines and radioactive elements Co OR Cs to do so

Question 18

What can depth filters be made of and what are they

Answer 18

They are a mesh of material overlapping in a random network and can be made up of paper or glass

Question 19

What can filtration be used for

Answer 19

Liquid or airflow
Liquids- resist clogging so can remove large particulates
Airflow- HEPA filters

Question 20

What is membrane filtration and what is it made off

Answer 20

It is a type of liquid sterilisation and is made up of hugh strength polymers such as nitro cellulose

Question 21

What does membrane filtration act like so what do they do

Answer 21

Act like a sieve so trap microorganisms on the filter

Question 22

What is chemical sterilisation used for

Answer 22

To sterilise surfaces e.g. 2% Trigene

Question 23

Where are microbes grown ( bacteria/fungi)

Answer 23

Grown in either liquid or solid media
Solid media- called agar
Liquid media- called broth
And can be grown in petri dishes or plastic/ glass bottles

Question 24

What is culture media

Answer 24

Mixture of nutrients in solution
Contains nutrients by microbes grown in lab
Contain buffer to maintain pH value
Liquid(broth) or solid(agar)

Question 25

What is agar and what is it used to make

Answer 25

It is a red algae polysacharide and is used to make solid media

Question 26

How to use agar

Answer 26

Ads 1.5-2% agar to broth and boil to dissolve agar Remains solid over incubation conditions 0-80 degrees and then compare with gelatin It liquifies at approx 100 degrees and gels at approx 42 degrees

Question 27

How does plate culture work

Answer 27

Spread sample over agar surface Bacteria grows on surface Sample places in plate and agar poured over it Bacteria grow within the agar Plates inverted before incubation and prevents condensation falling into plate

Question 28

What is defined media

Answer 28

It is the precise amount of nutrients I.e exact chemical composition And Highly purified inorganic salts and organic chemicals e.g. glucose and amino acids

Question 29

What is complex media

Answer 29

Rich in nutrients Chemically undefined (milk protein ,beef,yeast, soybeans) Support growth of microbes

Question 30

What is media formulated to be

Answer 30

General Selective Differential Enrichment

Question 31

What is general formulation and what materials does it use

Answer 31

Grow wide variety of bacteria | It used nutrient agar, yeast extract, meat extract, blood agar

Question 32

What does selective media include

Answer 32

Salt Bile Dyes e.g. crystal violet, malachite green Antibiotics e.g. penicillin, streptomycin Other chemicals

Question 33

What does staphylovoccus use (media)

Answer 33

Nutrient agar +7.5% NaCl Sodium deoxycholate which is used to inhibit gram postive skin bacteria in hand washing trials e.g. allows E coli k12 to grow

Question 34

What is mannitol salt agar used for

Answer 34

Media for the isolation of pathogenic staphylococci

Question 35

What is the ph indicator in mannitol salt agar

Answer 35

Phenol red

Question 36

What does coagulase postive and negative staphylococci produce

Answer 36

C positive s - produce yellow colonies due to mannitol fermentation C negative s - produce small pink or red colonies

Question 37

Differential media

Answer 37

Used to distinguish between one group and another Contains a chemical that is altered or utilised by certain microbes and not by others It is a form of biochemical test

Question 38

Chromagenic media

Answer 38

``` Contains substrates linked to chromogen (flurogen) Enzyme substrates (ONPG, CPRG, MUG) are organisms specific Results in colour change or flureosense under UV light ```

Question 39

Oxide UTI media

Answer 39

Two chromogens cleared by the B galactosidase and B glucosidase B gala... activity of E coli results in pink red colonies B glu.. activity of enterococci produces blue colonies Tryptophan deaminase activity produces a brown halo around colonies such as proteus sp

Question 40

What is enrichment media

Answer 40

Used to increase growth of particular microbe and inbit growth of others Used when organism is slow growing

Question 41

What does enrichment media rely on

Answer 41

Selective agent Specific nutrient Environmental conditions e.g. light and temp

Question 42

Example of enrichment media

Answer 42

Campylobacter C jejuni present in food in low number and causes food poisoning Need to enrich bacteria first can take several days Enrich using Bolton broth at 37 degrees, 4-6 hours, 41.5 degrees for 44 hours Incubate microaerobic at 41.5 degrees for 44 hours

Question 43

What are the incubation temp generally for pathogens

Answer 43

30 to 45 degrees

Question 44

Atmospheres for incubation

Answer 44

Aerobic Anaerobic Microaerophilic

Question 45

Microaerophilic/ aerobic incubation

Answer 45

Optimal growth conditions: 3 to 5% oxygen, 2 to 10% carbon dioxide Anaerobic need free oxygen enviro

Question 46

Ways of counting bacteria

Answer 46

Direct count Indirect methods Viable count

Question 47

Direct count

Answer 47

Number of cells are counted directly using microscope and chamber Numbers in known volume of culture is counted and then multiplied This is better for large cells e.g. yeast, fungal spores

Question 48

Viable count

Answer 48

Way of assessing growth of single celled organisms such as bacteria is the viable count Bacteria are grown on agar plate

Question 49

Plate count

Answer 49

Each colony formed from single bacteria | Count number of colonies you can determine number of bacteria present

Question 50

What is Dilutions used for

Answer 50

Sample with too many colonies can be diluted to ensure plates are countable. We need a plate between 30 and 300 colonies

Question 51

How does membrane filtration methods work

Answer 51

Membrane filter on a filter support Water sample filtered through membrane filter 0.45 um Membrane filter removed and places in plate containing the appropriate medium Incubation for 24 hours Typical colonies count etc

Question 52

What is membrane filter method useful for

Answer 52

Analysing aquatic samples or low number of organisms | And it can be combined with direct counting via fluorescent dyes

Question 53

What are Contact plates used for and how does it work

Answer 53

Used to fount number of bacteria present on surface Good for low numbers Plate places on surface and incubated then counted