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98 Cards in this Set
- Front
- Back
PURPOSE of Aseptic Technique and Culture Handling
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To learn how to handle cultures in an aseptic manner.
To learn how to inoculate various types of media and to identify various types of growth. |
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an ARTIFICIAL FORMULATION of nutrients and other essential substances required for microorganisms’ survival and reproduction
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Culture medium
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a culture medium that is LIQUID at room temperature
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Nutrient Broth (TSB)
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a medium containing PEPTONE and BEEF EXTRACT which is solidified with AGAR, a GELLING agent
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Nutrient Agar (slant) TSA
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a SPECIFIC MICROBE grown on a medium
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Pure culture
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the PROCESS by which microbes are TRANSFERRED from one medium to another using an inoculating instrument
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Subculturing
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Always incubate all bacterial plates in an _______ position.
This prevents ____________ from gathering on the ____ and dripping onto the ____ surface. |
inverted
condensation lid agar |
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Please stack the _____ plates in the ________ to conserve space and be sure to put your plates on the appropriate shelf for your lab section.
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Petri
incubators |
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When subculturing why must we use aseptic techniques?
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To insure the original stock and the new stock do not become contaminated.
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What is the first thing to do with the Petri plate?
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Label the bottom with a Sharpie.
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When pouring a Petri plate using molten agar in a tube, be sure to temper or cool the agar to _____ prior to pouring.
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~50 degrees
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When removing the cap drom the tube of agar what must you do with the cap?
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As soon as you remove the lid, discard the cap (This is the only time in Micro lab when it is OK to put test tube caps on your lab bench).
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What do you do with the lip of the tube?
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Flame it
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Why must you wipe off any water from the outside of the tube?
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This prevents contaminated water from dripping into your sterile Petri plate.
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Raise the lid of the petri plate, and pour the molten agar into the ________ of the dish while holding the Petri dish lid ______ the tube and plate bottom.
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bottom of the dish
above the tube |
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Holding the Petri dish lid above the tube and plate bottom prevents what?
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contaminants from falling into the freshly poured agar
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Leave the Petri plate stationary until _________.
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solidified
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What happens if you stack the plates?
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It will take longer for them to cool.
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Once solidified, the agar will have what kind of appearance?
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An opaque appearance.
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What must you do before removing a small sample of bacteria from the available culture?
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Sterilize an inoculating loop
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What must you do before you begin to streak the Petri plate?.
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Recap or close the culture once you have removed your sample.
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Lift the lid of the Petri plate. Streak quadrant #1 so that the bacteria are evenly spread. Then put the lid back onto the agar. (How must the lid be placed on the bench?)
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Never put the lid on the bench so that the inside of the lid faces the bench. The inside of the lid will pick up bacteria from the bench.
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After the first streak and after lid is put back on what is the next step?
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Flame the loop.
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After flaming loop, Touch one corner of quad. #1 pulling a sample into what quadrant?
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#2 and spread evenly
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What do you do after spreading sample into quad. #2?
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Flame the loop again.
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Repeat steps of flaming ____and removing sample from _______quadrants.
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loop
previous |
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You must flame the loop after each quadrant procedure ?
True or False? |
True
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The lid can stay off during each procedure?
True or False? |
False
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How can you est the coolness of your loop?
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You can test by touching the agar around the edge of the Petri plate. If it sizzles, its too hot. If it doesn't then it is ready.
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What is the goal of streaking a plate?
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To dilute out the number of bacteria you are moving with each additional streak.
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The division of individual cells
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Binary fission
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A population of clones that pile on top of each other and form this.
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Colony
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When you select a colony, you can be sure that you have generated a _____ ______.
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pure culture
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Why is the streak plate technique important when you have two unknown organisms?
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Because you want an isolated colony of each organism to make working stocks.
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What happens if you get a lot of moisture build up on nthe agar surface?
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You will not have colonies and you will generate a bacterial lawn.
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When oculating a slant, select bacteria using a ________ and use ___________.
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sterile loop
aeseptic technique |
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Flame the lip of the sterile agar slant keeping the cap in the crook of your ______ ______.
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pinkie finger
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Why must you not put cap down on the bench?
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Because you will pick up contaminents.
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Why must you always check the agar and broth cultures you are oculating?
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To insure there are no contaminating organisms already growing.
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Where do you begin when inoculating a slant?
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Begin at the bottom of the slant
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Touch the agar surface and make a _________ pattern across the agar surface moving _____ toward the opening of the tube.
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squiggle pattern
moving up toward |
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Be careful not to _______the agar surface.
When removing bacteria from an agar slant, do not ____ into the agar with the loop. |
do not to gouge
do not dig into |
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How should you label the slants?
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Slants should be labeled with your name, lab section, date, instructor's name and organism name.
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When working with aerobic or facultatively anaerobic organisms, keep the cap _______ ________ during incubation.
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slightly loose
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When Inoculating a Deep, use an __________ ___________..
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Inoculating needle
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If you do not have an inoculating needle, you can _________________?
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you can straighten out a loop.
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Flame the needle and remove a small amount of ____________ ________.
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bacterial growth
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Stab the ________ through the agar or gelatin. Go straight ___ and straight _____.
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inoculum
straight in and straight out |
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If your inoculating needle is too short, be careful not to stab the handle of the inoculating needle into the agar because ____?
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Because you have probably not sterilized the handle.
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After bacteria grow in an agar deep, you will see ______ along the _____ ______when you hold the tube up to the light.
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growth along the stab line
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If the organism is also _____ motile, you will see _____ ______ of the organism from the initial stab line throughout the agar.
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motile
outward motility |
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Is the pattern wider or narrower at the top of the tube?
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Wider
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What does this pattern represent?
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Inverted Christmas tree
since most of our organisms are aerobes and fstest growth will occur toward the top of the tube. |
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In gelatin, you will find that some bacteria are capable of breaking down protein in the gelatin resulting in ___________.
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Liquefaction
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Stabs should be labeled with what information?
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your name, lab section, date, instructor's name and organism name.
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When working with aerobic or facultatively anaerobic organisms, keep the cap ________ _________ during incubation.
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slightly loose
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When Inoculating a Liquid Broth, remove an inoculum of bacteria from a culture using a sterile ___________ ________.
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Inoculating loop
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What is the purpose of flaming the lip of the sterile broth culture and tilting the tube?
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so that the liquid can be reached with the inoculating loop w/o having to put the handle of the loop into the tube.
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What is the purpose of Twirling the loop around?
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so that bacteria are removed
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You will not be able to see the inoculum in the liquid unless_______?
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you have added too much.
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Another technique is to transfer the inoculum from ____ _____ to the _____ ______ of the test tube you are inoculating.
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from loop to the inside wall
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This technique will usually only work if ____?
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If the inside of the tube is not wet which means you should not shake or tilt the tube prior to this procedure.
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Once you have an inoculum stuck to the inside glass wall, you can tilt the tube so that _______?
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so that the bacteria are washed from the wall andf into the medium.
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What is the goal of this technique?
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To prevent introduction of the inoculating loop handle into the sterile tube.
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What is the purpose of heating the handle of an inoculating loop?
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to cut down on possible contamination
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Why must you not try to touch the handle of the loop against the mouth of the tube?
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This procedure will lead to contamination and spillage if the cap is not tight.
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Broth cultures should be labeled with what info?
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your name, lab section and organism name.
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An enteric organism; isolated from the intestines of animals.
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E. coli
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What are the colors of the colonies in E. coli?
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Beige and Mucoid
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Cells are very short bacilli with __________ _________.
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Peritrichous flagella
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Also enteric like the E. coli
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S. marcescens
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Colonies of S marcescens are what color?
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beige/pink at 37 degrees Celcius
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E.coli and S. marcesces are both ______________ pathogens.
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opportunistic
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organism found in soil and on skin of some animals
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M. leutus
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Is M. leutus a pathogen?
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NO
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Describe the colonies of M. leutus.
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Citron (sunshine) yellow
Have round margins and are raised (convex). Cells are cocci which occur in arrangements of tetrads. Non-motile. |
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Premade, sterilized and stored on the shelves at the front of the lab.
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TSB, TSA, slants and motility
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How do you melt deep TSA plates?
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In boiling water baths at the back of the lab.
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Agar melts at _______Degree celcius.
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100 degree celcius (boiling point)
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Once melted the deeps are transferred to the _______ holding tanks where they will be ________.
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50 degrees celcius holding tanks
cooled |
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Agar will solidify at what temperature?
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45 degrees Celcius
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TSB is inoculated with an ______ _______ and incubated at what degrees for E.coli and what degrees for M. luteus?
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inoculating loop
37 degrees celcius and 25 degree celcius |
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TSA plates are inoculated using ______ ________streak plate technique and incubated in an _________ position at temperatures as indicated above.
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four quadrant technique
inverted |
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Motility agar is _______ using an __________ needle and incubated with loose caps.
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stabbed
inoculating needle |
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TSA slants are ____________by ___________ the slant form bottom to top with bacteria usin and ________ ________.
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inoculated
streaking inoculating loop |
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Slants are incubated at what temperature?
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37 degrees celcius
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a general purpose liquid medium used to grow cultures
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TSB
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another word for turbid
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cloudy
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the ability of bacteria to move
(flagella) |
motility
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Relatively clear and allows you to visualize movement of bacteria through the agar in all directionsaway from the stab line.
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Motility agar
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smooth, complete coverage of the slant w/o contamination
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TSA Slants
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These slanta are perfect for storing large numvers of organisms on a relatively large surface that is sealable with a lid.
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TSA slants
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Cultures may be preserved for long periods of time in the refrigerator on ________.
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slants
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What produces a red pigment giving teh broth a red appearance?
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S. marcescens
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Usually are of a different color or texture and often do not lie on the streak line.
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Contaminants
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M. Luteus is motile or non-motile?
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nonmotile
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E.coli and S. marcescens are non motile or motile.
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motile.
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Wide Ribbon-like pattern of growth appears if _____?
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If you shake when inoculating the agar.
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