First lab Flashcards
Two organizations that put forth procedures and techniques to help prevent the spread of infectious diseases?
(CDC) Center for Disease Control and Prevention and (WHO) World Health Organization
A huge number of potentially preventable deaths occurred during the_______war.
Civil war
How do we disinfect our work place in the Microbiology lab?
We decontaminate the bench before and after lab period. Spray the desk with disinfectant, take a towel and distribute it over the table top and let it continue to dry on its own.
How did Dr. Lister (1800’s) disinfect his operating room?
With a diluted solution of carbolic acid (Phenol).
Do you place hazardous, infectious materials in the sink? This also includes solid material (this includes paper towel)
NO
How do you clean up spills of microbial cultures?
Cover the contaminated area w/ a paper towel that is soaked throughly w/ disinfectant. Let it remain on spill for ten minutes, then use paper towels to soak up spill. Towel goes in biohazard waste bag. Wash hands immediately afterwards.
What do you do w/ broken glass slides?
Don’t pick up w/ bare hands, sweep up the broken glass and place them in the specified container.
How do you dispose of glass slides and coverslips?
Wrap them in a paper towel and dispose of them in the biohazard container. Don’t discard demonstration slides.
Contaminated waste goes in biohazard bags, so where do non-biohazard waste belong?
Can be disposed of in appropriate containers, gloves can go in regular trash cans.
Where does inoculated media go?
To the 37 degree celsius incubator or room temperature storage tub, located in minilab (S213). We are responsible for proper disposal of all test tubes/petridishes in biohazard bags.
For short term storage of inoculated media?
Black refrigerators
When should you wash your hands thoroughly with soap and water?
Before class, after class, or when you spill something.
What are some good aseptic techniques to practice before class?
–Tie hair back –Wear a clean lab coat and gloves at all times –Decontaminate work bench before/after class –Wash hands throughly before/after class w/ soap/water
What are the five steps to hand washing?
Wet hands with water Apply hand wash (soap) Lather and wash for AT LEAST 15 seconds Rinse both sides of hands with water Dry hands and shut off faucet with towel
Two big groups of organisms found on your hands?
Resident and transient flora are on your hands.
Preventing pathogens from being transmitted from recognized and unrecognized sources?
Standard precautions includes the use of; hand washing, appropriate personal protective equipment such as gloves, gowns, masks, whenever touching or exposure to patients’ body fluids is anticipated.
If you are in an emergency situation, where hand washing isn’t possible what should you do?
Use an alcohol-based hand sanitizer.
What are nonsocomial disease and what is the number one way to prevent the spread of nonsocomial infection?
They are acquired in hospitals/healthcare facilities. To be classified as a nosocomial infection, the patient must have been admitted for reasons other than the infection. Some patients acquire nosocomial infections by interacting with other patients. Hospital staff can significantly reduce the number of cases with regular hand washing. They should also wear protective garments and gloves when working with patients.
What is an inoculating loop?
Allows the microbiologist to handle specimens in an aseptic manner. Has an aluminum handle and thin wire filament that rapidly heats up and cools down.
What does the Bunsen Burner and Incinerator do?
Bunsen Burner/Incinerator-connected by tubing to gas jet, ignited, and the flame is then used to sterilized the inoculating loop.
What should you do or not do after sterilizing your inoculating loop with the bunsen burner?
By inserting the inoculating loop in the hottest part of the flame you are using aseptic technique to sterilize it. Let the wire cool down for a few seconds before transferring your specimen from one medium to another. Do not wave in the air or blow on the inoculating loop.
What do sterile media consist of?
Nutrients for growing bacteria
What is the in vitro environment in which bacterial cells grow?
Medium
What is Agar?
A nutritionally inert substance that gives media its solid consistency. Must be heated to boiling to dissolve but will solidify at 40C. Can be allowed to solidify in tubes in an upright position, resulting in media termed stabs or deeps or in a slanted position producing slants w/ more surface area than stabs. Agar can also be poured to shallow dishes called Petri dishes to produce plates.
Definition of Sterile?
Material is free of all living forms.
What does the Autoclave do?
An autoclave is a pressure chamber used to sterilize equipment and supplies by subjecting them to high pressure saturated steam (moist heat) at 121 °C (249°F) for around 15–20 minutes depending on the size of the load and the contents. Kills even the most resistant endospores.
How should you label your media?
Use a sharpie to write your initials, the date, the type of medium, and the inoculum (specimen you are culturing) on the class of culture tube (never on lid) or the bottom side (w/agar) of the petri dish.
When working w/ media where should you place it at your work space?
Towards the middle, away from notebooks/books. Tubes should be placed in a test tube rack and petri dishes should be placed on top of the rack and secured w/ masking tape.
List the tube media.
TSB (Tryptic Soy Broth) TSD (Tryptic Soy Deep) TSS (Tryptic Soy Slant)
List plate media
TSA (Tryptic Soy Agar) TSP (Tryptic Soy Plate)
Aseptic vs. Sterile
Aseptic-free from contamination caused by harmful bacteria, viruses, or other microorganisms. (of surgical practice) aiming at the complete exclusion of harmful microorganisms. (of a wound, instrument, or dressing) surgically sterile or sterilized. Sterile-free from bacteria or other living microorganisms; totally clean.
How many Microbes are estimated by Scientist to be on earth?
5x10(30)
Microbes are ubiquitous (exist everywhere)? T or F ?
True
Can you explain why microbes are widespread?
1.) Their small sizes allow for easy dissemination by air and water. 2.) Demonstrate incredible versatility and flexibility w/ regards to their metabolism. 3.) Have a unique ability to tolerate extreme or unusual environments.
What do the microbes that live in oceanic and terrestrial environments do for earth?
The earth has limited quantities of carbon, nitrogen, and oxygen. Microbes are capable of recycling essential elements. Digesting the remains of once-living cells, returning basic chemicals back to the earth’s biogeochemical cycles.
Describe high species diversity (low numbers of existing species).
1.) Found in niches that do not contain many selective pressures. 2.) In soils and sea water Species diversity is the number of different species that are represented in a given community.
Define species richness (present in high numbers).
1.) Ecosystems w/ strong selective pressures inhibit the growth of all but a few species. 2.) Bacteria that can survive or tolerate the disinfectants used in hospital room. Species richness is the number of different species represented in an ecological community, landscape or region.
In what type of environments would you expect to find a large species diversity of microbes?
In niches that do not contain many selective pressures, such as soils and sea water.
How is it that your not sick all the time?
Our body provides us w/ adequate protection against microbial infections.
Given that niches w/ low selective pressures tend to have high species diversity, how do you think microbes are able to find nutrients and prevent other species from utilizing these nutrients?
Microbes demonstrate versatility/flexibility w/ regards to their metabolism, and have the ability to tolerate extreme environments.
What characteristics define extremophile?
Characteristics of extremophiles. Extremophiles are organisms that live in extreme conditions of temperature, acidity, salinity, pressure, or toxin concentration. Most extremophiles are single-celled micro-organisms belonging to two domains of life – bacteria and archaea.
Describe an extremophile.
Extremophiles are organisms that have been discovered on earth that survive in environments that were once thought not to be able to sustain life.
Why do the discoveries of extremophiles lead us to hypothesize that microbes may live elsewhere than Earth?
Possibility that life forms elsewhere might be a lot like the extremophiles that live in similar harsh conditions here on Earth. Microbes could live in the ice deep within comets, frozen there for eons until a collision with another planet or moon delivered them to a new home.
Synthetic media (defined media)?
Nutrient preparations used for microbial growth, the type of medium, exact composition and quantity of each nutrient is known. Used for fastidious microorganisms.
Complex or Chemically undefined media?
These types of media contain the same elements as defined media, but the amounts of each particular nutrient may vary from recipe to recipe.
What is Agar?
A polysaccharide solidifying agent derived from marine algae. Few organisms degrade it, it does not melt until its temperature reaches 95C and after being melted, it remains in a liquid state until it has cooled to about 40C.
What is an Agar Slant?
Liquefied medium that contains Agar and can be poured into a test tube or petri plate. The medium cools down and solidify’s at a slant.
What is Agar Deep?
Liquefied medium is allowed to solidify w/ the tube remaining upright.
What is an Agar Pour?
Medium that is poured into a large test tube and is intended for pouring into a petri plate at a later date.
What is an Agar Plate?
Liquefied medium poured into a petri plate in order to create a solid surface for growing bacteria.
Describe Dry-heat sterilization.
Used mostly for glassware, consist of a hot air oven at 160-170C for 2 hrs.
Describe Filtration
Used for media additives that cannot w/stand high heat sterilization methods. Liquid is passed through a filter that traps microorganisms while allowing the liquid to continue through the filter into a sterile container.
Describe Ultraviolet radiation.
Used in hospitals to sterilize the air or surfaces in a room.
Describe ethylene oxide.
A gas used to sterilize heat-sensitive materials, such as plastic petri dishes, cotton packing, and syringes.
What is the most important thing to remember about sterilization?
Materials cannot be 97% sterile-they are either sterile or not sterile.
List sterilization methods.
Autoclave, moist heat, dry-heat, filtration, ultraviolet radiation, ethylene oxide gas.
What is contamination?
The act of contaminating, or of making something impure or unsuitable by contact with something unclean, bad, etc. You can contaminate by your hands, the air you exhale, or inanimate objects. Microorganisms are everywhere.
What is aseptic technique?
Preserving the purity of objects.
How do you sterilize the inoculating loop?
Use the Bunsen burner to sterilize the inoculating needle by passing the base (closest to handle) through a flame to kill microorganisms. It must glow orange. Let the loop cool for several seconds. Do not set the loop down on the bench, don’t blow on it, do not touch it w/ your fingers to see if its still hot.
Why mustn’t you shake a tube of broth media?
The caps of the broth media have holes in them to allow for exchange of gasses. Shaking them will spill the media everywhere.
What to do w/ a cap when you take it off a test tube?
Caps remain on flask/tube any time the flask/tube is not in use. When you remove a cap you flame the mouth of the tube. The lip should be held at an angle to reduce the possibility that organisms in the air might fall into the tube. Never set the tube on the bench, remove the cap w/ the little finger of the hand holding the inoculating loop.
Describe pure culture.
Simply a culture of one type of organism.
What is inoculation (subculturing similar to inoculation)?
Microbiologist use this to transfer a pure culture of an organism. A tiny amount of microorganism is removed from one medium using an inoculating loop or an inoculating needle, and transferred or inoculated into or onto fresh medium.
Removing organisms from a liquid culture?
1.) Always grasp tubes by the glass portion, not the cap. 2.) Settling of the bacteria in broth tubes may have occurred. 3.) Gently shake or vortex broth tubes to resuspend organisms. Do not invert the tubes to mix the organisms (remember holes in the cap). 4.) Sterilize the inoculating loop, let it cool (10secs). 5.) Remove cap of the culture tube containing the inoculum w/ your little finger of the same hand that is holding the inoculating loop and hold the cap next to your palm. Flame the lip of the tube, two-three times, holding tube at an angle to minimize contamination. 6.) Stick cooled inoculating loop into the liquid bacterial culture. You should see liquid in the loop. 7.) Flame the lip of the tube containing the liquid culture. Replace the cap and return tube to the rack. 8.) Sterilize the loop.
What happens if your inoculating loop is to hot?
You’ll hear a hissing sound when you put it in liquid broth, which indicates that you are creating aerosols (steam), and you are also killing bacteria.
Removing organisms from slant culture?
1.) Sterilize loop and lip of test tube 2.) Insert loop into tube, scrape some of bacteria growing on surface of the medium using loop. Do not break the surface of the agar. A small amount of visible organism will suffice. 3.) Flame lip of the tube and transfer inoculum to fresh medium. 4.) Sterilize loop.
Removing organism from an Agar Plate Culture?
1.)Agar is contained in the bottom portion of the plate. Place the petri dish w/ the lid of the dish (big side) on the bench top. 2.) Sterilize loop. 3.) Pick up smaller bottom-side of petri dish that is filed w/ the agar w/ the other hand. 4.)Use loop to gently scrape a small amount of growth from the agar w/out gouging the agar. Tiny amount of organism is needed. 5.) Replace the bottom of petri dish on the lid and transfer the inoculum to fresh medium. 6.) Sterilize loop.
Why do we set the petridish upside down?
If you left it right-side up condensation would appear on the inside of the top lid and would drown the bacteria.
Inoculating broth medium?
Remove cap, flame lip of tube. Dip loop into broth, gently move loop back/forth to transfer organism from loop to the new medium. Flame neck of tube and replace cap.
Inoculating an agar slant?
Stab the loop down to the butt of the tube, bring loop back up the slant w/ a zig-zag motion, smear the rest of the inoculum on the slant portion of the agar. Flame neck of tube, replace cap.
Inoculating an agar deep?
Remove cap, sterilize lip of test tube. Stab loop 3/4 of the way to bottom of the deep, carefully w/draw loop. Do not need to move loop around b/c the mechanical action of stabbing the medium causes organisms to be transferred to the agar deep from the loop. Flame neck of tube, and cap it.
Inoculating an agar plate?
Place petri dish w/ the top of the dish (big side) on the bench top. While holding loop w/ the inoculum in one hand, pick up the smaller bottom side of the petri dish that is filled w/ the agar. With other hand gently streak the loop along the surface of the agar to transfer organisms. Without Breaking surface of medium.
Spot inoculation? (agar plate)
A concentrated area of organism about the size of a quarter is placed on the plate.
Streak-for-isolation-technique? (agar plate)
Bacteria are diluted across the surface of the medium in an attempt to isolate individual colonies of bacteria.
Why should a microbiologist wait until a liquid medium containing medium agar has cooled to 45C before adding living organisms?
The heat from the liquid could kill organisms. At this temperature the necessary nutrients can be added and it will have solidified.
Explain why you sterilize an inoculating loop? Before dipping into a tube?
Sterilizing the inoculating loop prevents contamination of the pure culture. And to sterilize after inoculation to kill/sterilize any microorganisms on the loop.
Explain why tubes should be kept as close to parallel as possible to the bench top while performing any transfers of inoculations?
To prevent airborne particles from contaminating the inside of the tube.
Most microbes cannot be grown by inoculating them into sterile water. Why? What must be provided for organisms to grow?
A standard carbon source is glucose, and nitrogen is often provided by peptones (partially digested proteins), or inorganic salts. Minerals and vitamins may also be provided, according to the growth requirements of the bacteria. Combinations of chemicals (buffers) may be used to keep the pH stable. Measured amounts of the concentrates are added to water, and dissolved to reconstitute the media.
What is a fomite?
An inanimate object or substance, such as clothing, furniture, or soap, that is capable of transmitting infectious organisms from one individual to another.
What is symbiosis?
Interaction between two different organisms living in close physical association, typically to the advantage of both.
What is a mixed culture?
Many types of microorganisms.
Pure culture?
Growth of only one type of microorganism in media w/out the presence of any other organism in that culture.
