Test Answers on Microbiology Lab Test 1
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TSA
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Tryptic soy agar
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What do you label your TSA plates with and where?
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Initials, date, class time and what was sampled; Bottom
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Scanning Objectives:
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4x: Scanning/Shortie 10x: low dry 40x: high dry 100x: oil immersion objective
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Tools used in lab:
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Kimwipes: used to clean stage and slides lens wipes: used to clean objective lenses ethanol: used to sanitize areas bibulous paper: used to blot dry stained slides inoculating loop/needle: used to collect samples
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Steps in storing a Microscope:
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1) lower stage 2) remove and clean slide with ethanol and kimwipes if oil was used. Place in slide box. 3) Scanning objective (shortie) facing stage 4) clean oil off the oil immersion objective (100x) with Lens paper and ethanol 5) clean the 40x objective as well, just incase it got oil on it. 6) Put dust cover back on and place in cabinet
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Bacteria are _________.
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everywhere
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We label plates with __________, __________, __________, and ________________________.
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Initials, date, class time 4:30, and what you put on plate
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We label plates _________________ so the view is not blocked.
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along the edge
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We use aseptic technique when we sample and work with bacteria. Examples are:
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-Open lid ever so slightly -heat-sterilize tools
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Aseptic Technique:
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free of pathogens or contamination
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We store plates _______________________, so that water from condensation during the incubation process will collect in the lid, not on the culture. It can be very messy, and can contaminate other samples.
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upside down/bottoms up
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Positive controls and negative controls _____________________________________.
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prove the process worked correctly
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What is a colony?
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A group of cells arising from a single parent cell.
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What is a CFU?
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Colony forming unit (name of parent cell)
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How can you tell different kinds of bacteria apart?
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shape, color, size
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How can you identify a fungal colony?
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fuzzy from hyphae
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If there do not seem to be any molds (fungi) why is it wrong to assume that ?
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Fungi grow at room temp after 5-7 days
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What are 4 shapes of bacteria?
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Vibrio, rod, coccus, spirochete
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Bacteria contain a cell wall made of __________.
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Peptidoglycan
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There are 4 objectives, ________________, ______________, ______________, and ____________.
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4x scanning, 10x low dry, 40x high dry, and 100x oil immersion
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The ocular piece has a lens with __________.
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10x
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Oil is only used with _________.
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100x
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_____________ is only used to clean the 100x lens, to clean slides, use ______________.
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lens paper; kimwipes
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Bacteria cells have 3 main shapes:
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-Cocci "o" -rods (Bacillus) -Spirochete
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Cell arrangement of COCCI can be in:
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-pairs (oo) -chains (oooo) -clusters
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Cell arrangement of BACILLI may be:
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-Single -Pairs -Chains
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Some cells have _________, which can be seen in the stained specimen.
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endospores ex: Bacillus sp. Clostridium sp.
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Terms:
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-Focus: Clarity -Parfocal: remaining in focus while increasing magnification of objectives. Just use the fine adjustment -Contrast: light and dark -Resolving power: the ability to distinguish two objects that are close together
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Ways to increase resolution:
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-blue filter -open diaphragm (max light) -raise condenser (max light) -oil for 100x lens, prevents light from defracting
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Calculating magnification:
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Total Magnification= ocular (10x) x objective 10x 4=40x 10=100x 40=400x 100= 1000x
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Dark-field and phase-contrast are used to observe protist and bacteria that are ______, staining cells ______ cells.
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alive; kills
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There are three types of movement observed with a microscope:
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-water current: caused by water evaporation -Brownian motion: caused by molecules hitting non-motile bacteria -true motility: caused by flagella
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brightfield:
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blue in/phase out
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darkfield:
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black in/phase out
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phase:
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blue in/phase in
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Dark field shows the _________ of specimen, and the field of view is _______ where there no objects are.
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outer surface; dark
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Phase-contrast shows the ________ structures, and the field of view is many shades of _______.
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integral; gray
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Why do we want to determine motility for bacteria?
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Identification
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The tube that has bacteria with motility looks _______, and the tunnel left by the stab is ________. In the tube with non-motile bacteria the tunnel left by the stab is __________.
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hazy(turbid); less visible; distinct
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Don't let tools sit in the __________ because they will burn you when you reach for them.
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Incinerator
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Don't wave _______________ to cool them down.
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tools in air
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Remember to use ______________ and open lid of culture partially.
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aseptic technique
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Compare agar and SIM (semi-solid media)
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-SIM is less concentrated, so more liquid. -Agar is solid. -Broth is a liquid
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Bacteria exist in _______ environments in mixed _________.
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normal; population
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Reasons to isolate bacteria:
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-Separate out bacteria from a mix -> pure culture -characterize bacteria (gram stain, shape, motility, size, cell arrangement) -Identification -antibiotic sensitivity test
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TSA (tryptic soy agar) is a _________ media which allows many different kinds of bacterial and fungal growth. EMB __________ is selective for gram (-) bacteria. That means that it does allow gram ____ to grow, but not gram ____.
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general; Eosin Methylene Blue; (-); (+)
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Notable colonies:
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TSA: red and white/cream EMB plate: green metallic sheen fish eye pink
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Smears should be ______, which means there is a small amount of bacteria rather than thick layers.
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thin
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Thick smears trap ______ and the many layers of cells prevent individual cells from being seen, therefore, smears should be ______.
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stain; thin
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There are three reasons we "heat-fix" a slide:
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1) kills bacteria 2) attaches bacteria to slide 3)retains size and shape of bacteria
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We have learned about three types of paper/tissue:
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-lens paper is for cleaning microscope objectives -kimwipes is for cleaning oil from slides and microscope stage -bibulous paper is for dying slides from stain
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Simple staining:
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-shows all cells present in smear -shows cell shape, size, arrangement
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The paper we use to dry stained slides is _______ paper.
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bibulous
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Methylene blue contains a ___________, a charged colored cation (+), which attaches to negatively charged _______________.
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chromophore; protein & DNA
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Gram-staining is very useful, because it can show you:
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-cell wall position -cell shape and arrangement
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The purple crystal violet stain is held in the thick layer of _________________ in gram positive cells.
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peptidoglycan PURPLE-POSITIVE-PEPTIDOGLYCAN
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The step that requires the most skill and attending is the _______________ with alcohol. If you leave the decolorizer on too long, the crystal violet will wash out of the thick peptidoglycan layer of the gram (+), as well as the thin layer of peptidoglycan in gram (-). Then all bacteria will appear gram (-).
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decolorizer
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Also, if you do not decolorize long enough, the crystal violet will remain in the thin layer of peptidoglycan of gram (-). Then all bacteria will appear gram ____.
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(+)
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Steps of gram staining:
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(After heat-fixing) -crystal violet x20 seconds -wash with water x2 seconds -Gram's Iodine x1 minute -Decolorize with alcohol x10-20 seconds -wash with water x2 seconds -Safranini x1 minute -wash with water x2 seconds -blot dry with bibulous paper Used: Staphylococcus aureus and Pseudomonas aeruginosa
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Gram (+) appears _____.
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purple
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Gram (-) appears ______.
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pink
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Some bacteria cannot be diagnostically stained by the gram stain. They have a high amount of waxy material called ______________.
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Mycolic acid
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Two examples of bacteria that have mycolic acid are ________________ and __________________.
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-Mycobacterium sp. -Nocardia sp.
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The ____________ stain is a diagnostic tool for the identification of Mycobacterium ____________ and Mycobacterium ______________ (causing the disease leprosy).
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acid-fast; tuberculosis; leprae
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Staphylococcus aureas:
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gram (+), coccus non-acid fast, cocci
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Pseudomonas aeruginosa:
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gram (-), rod non-acid fast, rod
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Mycobacterium phlei:
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acid fast (+), rod
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Bacillus subtilis:
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non-acid fast, rod contains endospores
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Acid-fast staining steps:
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(After heat-fixing) -Carbolfuchsin x5 minutes -wash with water -Decolorize with acid alcohol x1 minute -wash with water -Methylene blue x30 seconds -wash with water -blot dry with bibulous paper
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Acid-fast stain appears _______.
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fuschia
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Non acid-fast appears ______.
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blue
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In tube culture method:
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Staphylococcus epidermidis: no motility; Bacteria grew only where inoculated, leaving a distinct tunnel. Psuedomonas aeruginosa: motility; Bacteria with flagella are able to swim away from the stab made by the inoculating tool. Agar will appear hazy, with no distinct tunnel.
