Ex. 1 - Aseptic Techniques and Inoculation of Media Flashcards
There are accepted methods of carrying out activities or operations in a laboratory where pathogenic or non-pathogenic microorganisms are handled.
Good laboratory practices
This can also help in minimizing variability, and improve reliability and reproducibility of microbiological data that is obtained.
Careful oberservance
This is the basic component of most microbiological tests.
Culture media
T or F: It is important to choose the appropriate media or components in making the media based on accepted references.
True
T or F: Different media types have different media requirements (e.g., heating, additives, pH adjustment)
True
It is the universal diluent for microbiological media.
Water
Type of water is the most often used in media preparation.
Purified water (NOTE: in some cases, deionized or distilled water can be used depending on the instructions)
T or F: Consistent preparation of media DOES NOT require accurate weighing of dehydrated media or media constituents.
False - it is required
T or F: Dehydrated media should be thoroughly dissolved before dispensing and sterilization.
True
What is the general indication of overheating?
Darkening of media or Maillard-type reaction or Nonenzymatic browning
T or F: Sterilization of media can be performed even without parameters from the manufacturer or validation from the user.
False - it should be performed within the parameters provided as well as based on the validity of the user
What is the most preferred sterilization technique? What is the catch?
Autoclaving with moist heat, it cannot be used for heat-labile components as it may deteriorate and harm the quality of the media
The second appropriate sterilization technique that can be used
Sterilization by filtering or filtration
Recommended parameters when using an autoclave
121 degrees Celsius for 15 minutes at 103 kPa (15 psi)
T or F: Sterilization time will be dependent on the media volume and autoclave load.
True
T or F: Storage of media in the autoclave after the liquid cycle is completed is recommended.
False - it is not recommended as it may damage the media
How many times should re-melting of media be done?
Only once
If re-melting will be done, how should it be done?
Should be done in a heated water bath or by using free-flowing steam
In microbiological cultures, what should be done before use in quality control testing?
Confirmation of the purity of the culture and its identity
This technique is the one recommended for storage of stock cultures.
Seed-lot technique
T or F: Discard unused portion of cell suspensions to minimize the risk of viability and contamination of the stock.
True
T or F: The number of transfers of working control culture should be tracked.
True - to prevent excessive sub-culturing that increases the risk of phenotypic alteration or mutation
T or F: The laboratory layout and design should minimize cross-contamination of microbial cultures.
True
A laboratory should be divided into ____?
Two, clean or aseptic areas and live culture areas
Equipment that are difficult to sanitize (e.g., refrigerators and incubators) should be dedicated to _______ to minimize potential of inadvertent contamination.
aseptic operations and live culture operations
T or F: All microbiological samples should be taken using aseptic techniques, including nonsterile products.
True
Is it important to minimize the time between sampling and initiation of testing?
True - to control storage conditions
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.
Aseptic Techniques
Examples of aseptic techniques
- 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
T or F: People should only wear lab coats that are worn only in the laboratory.
True
Bench tops and shelves should be washed immediately before all uses with _____? And what could be an alternate?
10% v/v household bleach containing sodium hypochlorite
Solution of antiseptic cleanser (e.g., Lysol)
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.
True
Give examples of reagents
Antibiotics, drugs, sugars, amino acids, vitamins, and complex media
What could physically remove living organisms in preparing media?
A range of syringe-based or bottle-top filters of different pore sizes
Filtration may use what type of pressure?
Positive and negative (vacuum) pressure
T or F: Negative pressure is more advantageous as it reduces clogging from precipitated salts.
False - positive pressure
Filtering procedures are usually carried out in what way?
In a laminar flow unit or on a bench equipped with a Bunsen burner to avoid recontamination of just-sterilized materials
Most living organisms are retained in what size of filter?
0.45 micrometer filter
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?
0.22 micrometer filter
Autoclaving means _____
Using steam under pressure
It is a rapid method of sterilizing almost anything except heat-labile substances
Autoclaving
T or F: The typical autoclaving conditions are sufficient to kill virtually all forms of life, including bacterial endospores, and will inactivate viruses.
True
What are examples of objects that will be autoclaved longer due to having a slower surface heating or steam penetration?
Large bottle of media, bulky bag of biohazard waste, or a beaker full of microcentrifuge tubes with aluminum foil covering
What are the substances that cannot be autoclaved under any circumstance?
Volatile solvents or corrosive chemicals (e.g., phenol, trichloroacetic acid, ether, chloroform), or any radioactive isotopes
Items that can undergo oven sterilization
Glassware, metal, and objects that will not melt at a temperature between 121 and 170 degrees Celsius
Typical conditions for oven sterilization
160 degrees Celsius for less than or equal to 2 hours and 170 degrees Celsius for 1 hour
It is a method of choice for the treatment of glassware used for work with ribonucleic acid (RNA).
Oven sterilization
T or F: Any aseptic work should be completed as quickly as is comfortable to minimize the risk of contamination.
True
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.
Open flame
The ______ or _______ can be used to sterilize inoculating loops, warming glass bottle necks, or igniting alcohol on culture spreaders.
Bunsen burner or alcohol lamp
Apparatus that uses infrared heat at a temperature of 815 degrees Celsius to sterilize the wire portion of the inoculating loop.
Micoincinerators
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.
Laminar flow unit or hood
What is the most important part of the Laminar flow unit?
High-efficiency bacteria retentive filters such as the HEPA (high-efficiency particulate air) filter
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.
True
What are the two flow hood design types?
Horizontal and vertical
Give the major concept of the laminar flow unit.
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.
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.
True
What is the purpose of flaming?
To warm the opening and create air convection currents up and away from the opening to help prevent the entrance of dust particles
How should the medium be arranged in petri dishes?
Should be arranged on the lab bench convenient to the burner or microincinerator
What is used when individual colonies of bacteria or fungi, growing on a solid surface, are required?
Agar culture
Agar culture inoculation method wherein a loop is used to well isolate individual colonies that can be detected on the parts of the plate.
Streak plate dilution technique
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.
Lawn technique
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.
Stab inoculation
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.
Point inoculation
Agar culture inoculation method where it is inoculated across the top with a ‘wiggly’ line up to the surface of the slant.
Inoculating slants
T or F: Broth cultures are used where individual colonies of bacteria of fungi, growing on a solid surface, are required.
False - not required
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.
True
