Ex. 1 - Aseptic Techniques and Inoculation of Media

Question 1

There are accepted methods of carrying out activities or operations in a laboratory where pathogenic or non-pathogenic microorganisms are handled.

Answer 1

Good laboratory practices

Question 2

This can also help in minimizing variability, and improve reliability and reproducibility of microbiological data that is obtained.

Answer 2

Careful oberservance

Question 3

This is the basic component of most microbiological tests.

Answer 3

Culture media

Question 4

T or F: It is important to choose the appropriate media or components in making the media based on accepted references.

Answer 4

True

Question 5

T or F: Different media types have different media requirements (e.g., heating, additives, pH adjustment)

Answer 5

True

Question 6

It is the universal diluent for microbiological media.

Answer 6

Water

Question 7

Type of water is the most often used in media preparation.

Answer 7

Purified water (NOTE: in some cases, deionized or distilled water can be used depending on the instructions)

Question 8

T or F: Consistent preparation of media DOES NOT require accurate weighing of dehydrated media or media constituents.

Answer 8

False - it is required

Question 9

T or F: Dehydrated media should be thoroughly dissolved before dispensing and sterilization.

Answer 9

True

Question 10

What is the general indication of overheating?

Answer 10

Darkening of media or Maillard-type reaction or Nonenzymatic browning

Question 11

T or F: Sterilization of media can be performed even without parameters from the manufacturer or validation from the user.

Answer 11

False - it should be performed within the parameters provided as well as based on the validity of the user

Question 12

What is the most preferred sterilization technique? What is the catch?

Answer 12

Autoclaving with moist heat, it cannot be used for heat-labile components as it may deteriorate and harm the quality of the media

Question 13

The second appropriate sterilization technique that can be used

Answer 13

Sterilization by filtering or filtration

Question 14

Recommended parameters when using an autoclave

Answer 14

121 degrees Celsius for 15 minutes at 103 kPa (15 psi)

Question 15

T or F: Sterilization time will be dependent on the media volume and autoclave load.

Answer 15

True

Question 16

T or F: Storage of media in the autoclave after the liquid cycle is completed is recommended.

Answer 16

False - it is not recommended as it may damage the media

Question 17

How many times should re-melting of media be done?

Answer 17

Only once

Question 18

If re-melting will be done, how should it be done?

Answer 18

Should be done in a heated water bath or by using free-flowing steam

Question 19

In microbiological cultures, what should be done before use in quality control testing?

Answer 19

Confirmation of the purity of the culture and its identity

Question 20

This technique is the one recommended for storage of stock cultures.

Answer 20

Seed-lot technique

Question 21

T or F: Discard unused portion of cell suspensions to minimize the risk of viability and contamination of the stock.

Answer 21

True

Question 22

T or F: The number of transfers of working control culture should be tracked.

Answer 22

True - to prevent excessive sub-culturing that increases the risk of phenotypic alteration or mutation

Question 23

T or F: The laboratory layout and design should minimize cross-contamination of microbial cultures.

Answer 23

True

Question 24

A laboratory should be divided into ____?

Answer 24

Two, clean or aseptic areas and live culture areas

Question 25

Equipment that are difficult to sanitize (e.g., refrigerators and incubators) should be dedicated to _______ to minimize potential of inadvertent contamination.

Answer 25

aseptic operations and live culture operations

Question 26

T or F: All microbiological samples should be taken using aseptic techniques, including nonsterile products.

Answer 26

True

Question 27

Is it important to minimize the time between sampling and initiation of testing?

Answer 27

True - to control storage conditions

Question 28

This is a set of routine measures that are taken to prevent cultures, sterile media stocks, and other solutions from being contaminated by unwanted microorganisms.

Answer 28

Aseptic Techniques

Question 29

Examples of aseptic techniques

Answer 29

- Cleaning and disinfecting lab surfaces - Limiting the duration that cultures or media are uncapped or exposed - Keeping petri dishes closed whenever possible - Effectively sterilizing inoculating loops and other equipment that comes into contact with cultures or media - Avoiding breathing on cultures or sterile instruments

Question 30

T or F: People should only wear lab coats that are worn only in the laboratory.

Answer 30

True

Question 31

Bench tops and shelves should be washed immediately before all uses with _____? And what could be an alternate?

Answer 31

10% v/v household bleach containing sodium hypochlorite Solution of antiseptic cleanser (e.g., Lysol)

Question 32

T or F: Organic disinfectants such as 70% v/v ethanol are generally less effective than bleach solutions since ethanol are volatile and may evaporate too quickly and may not completely inactivate all potential contaminants.

Answer 32

True

Question 33

Give examples of reagents

Answer 33

Antibiotics, drugs, sugars, amino acids, vitamins, and complex media

Question 34

What could physically remove living organisms in preparing media?

Answer 34

A range of syringe-based or bottle-top filters of different pore sizes

Question 35

Filtration may use what type of pressure?

Answer 35

Positive and negative (vacuum) pressure

Question 36

T or F: Negative pressure is more advantageous as it reduces clogging from precipitated salts.

Answer 36

False - positive pressure

Question 37

Filtering procedures are usually carried out in what way?

Answer 37

In a laminar flow unit or on a bench equipped with a Bunsen burner to avoid recontamination of just-sterilized materials

Question 38

Most living organisms are retained in what size of filter?

Answer 38

0.45 micrometer filter

Question 39

However, bacteria can still pass through despite crossing into the 0.45 micrometer filter. What size of filters should then be used to ensure sterilization of the fluids?

Answer 39

0.22 micrometer filter

Question 40

Autoclaving means _____

Answer 40

Using steam under pressure

Question 41

It is a rapid method of sterilizing almost anything except heat-labile substances

Answer 41

Autoclaving

Question 42

T or F: The typical autoclaving conditions are sufficient to kill virtually all forms of life, including bacterial endospores, and will inactivate viruses.

Answer 42

True

Question 43

What are examples of objects that will be autoclaved longer due to having a slower surface heating or steam penetration?

Answer 43

Large bottle of media, bulky bag of biohazard waste, or a beaker full of microcentrifuge tubes with aluminum foil covering

Question 44

What are the substances that cannot be autoclaved under any circumstance?

Answer 44

Volatile solvents or corrosive chemicals (e.g., phenol, trichloroacetic acid, ether, chloroform), or any radioactive isotopes

Question 45

Items that can undergo oven sterilization

Answer 45

Glassware, metal, and objects that will not melt at a temperature between 121 and 170 degrees Celsius

Question 46

Typical conditions for oven sterilization

Answer 46

160 degrees Celsius for less than or equal to 2 hours and 170 degrees Celsius for 1 hour

Question 47

It is a method of choice for the treatment of glassware used for work with ribonucleic acid (RNA).

Answer 47

Oven sterilization

Question 48

T or F: Any aseptic work should be completed as quickly as is comfortable to minimize the risk of contamination.

Answer 48

True

Question 49

Aseptic technique used to create a cone of hot air above and around the laboratory bench to reduce the viability of organisms on suspended dust particles.

Answer 49

Open flame

Question 50

The ______ or _______ can be used to sterilize inoculating loops, warming glass bottle necks, or igniting alcohol on culture spreaders.

Answer 50

Bunsen burner or alcohol lamp

Question 51

Apparatus that uses infrared heat at a temperature of 815 degrees Celsius to sterilize the wire portion of the inoculating loop.

Answer 51

Micoincinerators

Question 52

This apparatus provides the workspace with clean, ultra-filtered air, keeping room air from entering the work area as well as suspending and removing airborne contaminants.

Answer 52

Laminar flow unit or hood

Question 53

What is the most important part of the Laminar flow unit?

Answer 53

High-efficiency bacteria retentive filters such as the HEPA (high-efficiency particulate air) filter

Question 54

T or F: The laminar flow unit captures a minimum of 99.97% of dust, pollen, mold, bacteria, and any other airborne particles with a size of 0.3 micrometers.

Answer 54

True

Question 55

What are the two flow hood design types?

Answer 55

Horizontal and vertical

Question 56

Give the major concept of the laminar flow unit.

Answer 56

Room air is taken inside the unit and passed through a prefilter, removing gross particles of contaminants. Then, the air is compressed and channeled to the HEPA filter. Lastly, the purified air will flow out over the entire workplace or work surface in parallel layers at a uniform velocity with no disruption between layers.

Question 57

T or F: All growth media, cultures, and sterile reagents should be manipulated inside the cone of heat that is created above and around an open flame or within a laminar flow hood.

Answer 57

True

Question 58

What is the purpose of flaming?

Answer 58

To warm the opening and create air convection currents up and away from the opening to help prevent the entrance of dust particles

Question 59

How should the medium be arranged in petri dishes?

Answer 59

Should be arranged on the lab bench convenient to the burner or microincinerator

Question 60

What is used when individual colonies of bacteria or fungi, growing on a solid surface, are required?

Answer 60

Agar culture

Question 61

Agar culture inoculation method wherein a loop is used to well isolate individual colonies that can be detected on the parts of the plate.

Answer 61

Streak plate dilution technique

Question 62

Agar culture inoculation method wherein an aliquot of liquid sample is spread across the surface of the agar plate to make one continuous 'lawn' of bacteria.

Answer 62

Lawn technique

Question 63

Agar culture inoculation method where in a liquid sample is mixed in with molten agar as the agar is poured into the plate. This has an advantage compared to streak plate since it allows microbes to be evenly dispersed throughout the agar.

Answer 63

Stab inoculation

Question 64

Agar culture inoculation method wherein plates are inoculated with filamentous fungi (molds). The plate is also held upside down and the wire is pushed upwards to prevent the spores from falling off the wire.

Answer 64

Point inoculation

Question 65

Agar culture inoculation method where it is inoculated across the top with a 'wiggly' line up to the surface of the slant.

Answer 65

Inoculating slants

Question 66

T or F: Broth cultures are used where individual colonies of bacteria of fungi, growing on a solid surface, are required.

Answer 66

False - not required

Question 67

T or F: Liquid transfers mix in readily while solid transfers need to be 'swirled' into the broth to transfer the solid material into the broth.

Answer 67

True