LAB GEN MICROBIOLOGY
LAB GEN MICROBIOLOGY BIOL 208
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This 15 page Class Notes was uploaded by Sally Jast on Wednesday September 30, 2015. The Class Notes belongs to BIOL 208 at Western Kentucky University taught by Staff in Fall. Since its upload, it has received 26 views. For similar materials see /class/216722/biol-208-western-kentucky-university in Biology at Western Kentucky University.
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Date Created: 09/30/15
208 MICROBIOLOGY SECTION 1 e omnTA nou ma nous AND mum Asgcmmns m 5mm 113115 1 m mcnoscop euzm z mumqu AND xmmmc cums Mmys wear a elem we em a appmVEd Maharame aumrmxe whm mkag a me bmth Yam Instmcmxwmcammeman we mummy 2 Ways we app aved eye pmeeuan when summg ax mmmg hazaxdaus Maharame ehemmxs ma reagems 3 Ways read rheumame altexuses pnax m yam sehe Maharame meeung 4 A me Sun me me at each Maharame 555mm smdenws shauld dean max assigned bmemap Mm a msmfetmmsalunan pmmea s Phteanly nscssary menus an me ummaxy bench mp CDMS mxacases ma bank bugs my be m m mm afzhe Maharame Dr M the aar by me cannck 5 an REP Humans xmmmmy Ifyau shauld spm ax map a eumue 1 15 m be Cwexed eampxenexy wxrh dlsmfetmm salunan Clearly wee a Fem mshes tubs ma uid A Culture vessels Mus be label d Mm W and henmeumemme matuhmd when r1115 15 mm eath studenzshauld purchase a rm pamzshnpi pamanmz mukEX SAFETY and GENERAL LABORATORY39 HRECTTONS CONTINUED 8 Do not place contaminated materials on the bench top or in the trash LOCATE ALL HAZARDOUS WASTE CONTAINERS IN THE LABORATORY eg sharps pipets contaminated glasswaremedia contaminated disposable materials 9 Smoking drinking and eating are prohibited On no account should any apparatus or materials used in the laboratory be placed in the mouth 10 Do not wear jewelry to laboratory sessions and cover all navel piercings while working in the laboratory 11 Restrain long hair from hanging over cultures or Bunsen burners Bunsen burners must be turned off when not in use and when ammable solvents are in use nearby 12 Microbial cultures are to be opened and manipulated only in the vicinity of a Bunsen burner with a strong ame Under no circumstances should culture material be touched directly with any part of your body use inoculating loops pipettes etc to transfer cultures 13 All cuts must be reported to the instructor and adequately covered before entering the laboratory Latex gloves will be provided at the front desk 14 Develop a safety firstquot attitude to working with cultures do not take risks and never hesitate to ask questions when you are in doubt 15 Keep all cell phones and electronic devices on silent and in your purse or book bag Do not handle electronic devices while working in the laboratory 16 Always wash and dry your hands before leaving the laboratory HEALTH If you are pregnant or planning on becoming pregnant If you suffer from any disability such as epilepsy diabetes or a heart condition if you have serious allergies a known infection or are being treated with immunosuppressive drugs such as corticosteroids or if you are colorblind please tell your class supervisor The information will be kept entirely confidential 12 W5 Each student should know the Lucauun of the nearest re alarm pull ca of the Lucauun of the Fun Am kn Lucauun ufzhe eyemsh Szauun IMPORTANT PHONE NUMEE Emergency Police em mummy Police Services DE ares re mmgmsher R ESF111 and Rest 27077 7 a us 911 WKU s 45 254 mam campus TMENT 0F BIOLOGY OFFICE 270774573 96 WKU mam campus Environmental Health and Safety 270774575973 WKLV mam campus min 15 A Tar PRIORITY AND lenus DISCIPLINI n AGE AND IVIIY s s39rA39nNG TH TUDlNT INSTRUCT CONDUCT LABORATORY IX ERUSIS YOU WILL In HOME TO 51m A nocumm39 on HA COVIRED THI lawnon sunquot amnums AH39IRWHICH mu Au cuAu Au AIOVE D o Exerclse L W A base mlcmscape Cansms at Ma le 55 ma me Essatumd hardware m make mg at speemlens sasl x The upp mast lms 51126 me scum ls me pm rhmugh whth a pexsan mks The law 1211515 me a lee Eva a we Enwwmg npm Changmg at slneeme lmses The may lube abletnve lmses m plsee Mas mcmmal glml speelmms are named n g as sums ma plsem an me 5mge FIGURE 1 1 OPTICALAND MECHANICAL FEATURES OF THE COMPOUND MICROSCOPE ocular lense e revolving nusepiece oblectlve lenses 13 course focus specimen stage line focus condenser 39 39 V luminam mechanlcal stage controls illumination control power swilch Usualle Cups ax emps ham me shde may m me 5mge Yam mmmstape ls mulpp d wlrh mechanmu 5mge nch me msuutmx wul aemsnsune me nape use ma funcnan at r1115 feature unmmn a rheawhr by m magm umn at me ablscn As light passes through various substances glass air specimens etc it bends This bending of light is called refraction The refractive index of a substance is a measurement of the extent that the substance bends light Excessive refraction can cause distortion of the image At magnifications of less than 400x the distortion is minimal But at higher magnifications the distortion becomes so great that image details are lost An oil immersion lens helps to remedy this problem by eliminating the air gap between the specimen and the objective lens A drop of special immersion oil is placed on the microscope slide and the oil immersion objective lens is maneuvered so that it is touching the oil Immersion oil has the same refractive index as glass so that the light passes through the slide specimen oil and objective lens as if they were a single piece of glass The instructor will illustrate the use of immersion oil Resolution is the ability to tell two points apart as separate points If the resolving power of your lens is 02ym that means two points that are 02ym apart can be seen as separate points If they are closer than 02ym they will blend together into one point To calculate the Resolving Power RP of a microscope you must know the wavelength of light A and the Numerical Aperture na of the objective lens usually found on the objective eg 100X Lens 12 S RP 702 x na FOCUSING THE MICROSCOPE Prepared Slide Candida albicans This is a stained smear of a unicellular yeast the purpose of this exercise is to learn and to practice the proper use of the microscope PROCEDURE Step 1 Obtain a prepared slide Mount the slide onto the stage of the microscope and bring the stage to its lowest position Step 2 Start with the 10X objective in place Do not look into the ocular lens at this point Using the course adjustment knob and watching from the side of the microscope move the stage with specimen to its highest position assuring it does not contact the objective lens Now you may look through the ocular and focus downward with the coarse adjustment until an image comes into view Use the fine adjustment to obtain maximum clarity From this point on do not use the coarse adjustment doing so can result in damage to the lens slide or both Adjust the iris to allow enough light for maximum visibility and contrast Usually this will be about half the maximum iris opening Too much light can wash out the details of the image Step 3 Move the slide to a point of interest Move the next objective lens into place 40X and adjust the fine focusing knob and adjust the iris as necessary Your instructor my ask that you go directly to the 100x oil immersion lens skipping the 40x objective Step 4 Note that as the power of the objective lens increases the distance between the objective and the specimen working distance decreases Also as magnification increases the field of view visible area and depth of eldfocus visible thickness decrease Moving the fine adjustment up and down allows viewing of other areas along the depth of thickness of the spec1men 15 sue 5 Ta use me ammmesmn 1m Do or msan nu focus mwe me mnemmm between me 40x abyemve mean 12115 me me 100x DMscme ml lens mee a map at Immexsmn ml enemy an me slide Mave me ammmemn 12115 mm puee ma mam me ne me N 121155 Make me at me dx aentes ma Mame between me axgmms The msuucmxwm assslyau m r1115 plateau Color pines llL2l3 Mi w hlylulu Sn L The mm um be p ated m an me shelf m me 4 Pasman Suth mmhe 4x mung lms 5 pmeeung numde Mm me xascape 51m mm quPEd setuxely mum me mu me me meemmm Smge rank e mpl mlyxelnum 14x ahieem I an em Illal is u w W ocum Luvs omcnv Luvs ms mmmcm counmsm mm anch39noN womuc mmucz OIL IMMERSION RESO LVING POWER H Why do we use immersion oil with the 100X objective N What is the function of the Iris diaphragm LN What is the advantage of the parfocal lenses 4 The ocular lens on your microscope is 10X If a 5X ocular were used with the same three objective lenses ie 10X 40X and 100X what magnification would be achieved 5 How would a decrease in the wavelength of light effect the Resolving Power Remember RP MZ X na 17 Exams 2 W WA Mi mm s me mm mm Mum Ts mm mans m M mmmgsmsms m mm mm Culture medium 111 mean Culture mm are salunans Canmmlng all at me mums ma netssary physm gmwm pummemxs nscssary m mxcmbul gmwrh are what m an mmmargamsms can gmwm any g 11 culture medium m m m many cm gmw In my knawn culmxe medium m maman m Ch mul ma physml Chantmnsnts mm Can be msmgmshm Qualmnvely as sand ax mm mm mum1 A mam 15 a mum gmth media Mas cultures In r1115 Maharame wm be pxwmded m a mum 1126 51 Num m md39i39 The msuucmx W1 mscuss r112 mgredl us ma r112 Spetul prepmnan am mum means W agar Sand medium 15 media Canmmmg lnr39 ax same ama masdy men submiymg agenn Sand medium ms physical sumuxe mam lurks summex ma r1115 allaws when m gmw m physmny mfuxmauve ax usammys leg as Calam sl Sand medium 15 uslu y used as 1 Slams 2 smhs a Flaws pem msms Mn npnIwahmdkdnvd mldl kxwmduim WNWdWlwhumnu W m sums mans vaded an Nuuxml Am sums lane an eath we Mmmms mm vaded anuummmmnms lane an eath we W howlI lunleltllatl m slut nlmu Each studentwm label ane Nument mm m are m Wm yam nun mum ma the mm m swans mam mm a sharpie atyautmx mmmg pmct ms wulmfmm Wane mm m mnuuwnh an mm mu 1 beinngs m yau mu m y u have maeuhtm u wtthsmunsnnans mm labeling ts saier guldehnex 2 Inezhm lumutAm sum an ill In Each studentwm label ane umem gushnt as amve mmtmned 2 Incline mantax stun mm pm until Each studentwm label a sscand u unlm my sum as abwe mennaned yam plate culture 15 Mmmm mm The msnutmtwm demansuam ASEpu teehnmue W NOTE All tub ultun m m b in suppomu quota in u upribe pan39u39an at in your hunt NEVER rua cuntu man FLAT mu amenmp IURFAGI BERCISE 2A Inuulm Nutrient Bran mm shun culture 433 sway 11m Loor 7 72 Ham the matuhnng lamp like a palm and 112 the wue m u am My pm mm mm mm mm m m w m My my 5 We be 2me mmm 4 R by mm my m 2 on m a m m o xlocvmu ll dunnumb um um hme mg a mg mm mm m mm W gmp my mm m be 1m Mm mm m m Mmym mg mm mm m m an mg mp my mmmmm m 5 mm m m o m m or woman may pass m up We be mm Mum am COLLECTINOCULUM Plate the stetuelaap mm the baCEHAI culture and tmave a 5mm maumrmmm m ch39nvA39r 39ru m r mm m mummy mm m 41m m up a m m or mome may pass m up We be mm Mum mm mm um mg m pm mmmwmmwm my m m quotme my sun 5 cm 1 un or 11 mm 1AllLIV39 Mmquot non um r112 mus nger at me ma haldmg r112 umsfex lawn grasp ma XEmDVE r112 lube 1m Cannnue ham me ha w hyaux mug ngEl39 mung me m an r112 mbl mp nsks mmmmmn my a mu n up or n snmz 1 sz lumm39r non ughuy pass me up mm lube rhmu h rhebumex mme Twsmmocuwm Plate r112 smx elaap mm me ummz mfh E2 mm m m plate me mm mm map mm r112 smug mm m 5mm ham r112 lube Man mgle m ama m e 2 hr m mmmmm msuu wm demansuam r1115 Ermqu my 7 mm n up or n snmz 39LAB39ILIV39 lVTlllllT non ughuy pass me up mm lube rhmugh rhebumex mme befme dasmg r112 tuba Plate r112 matuhzm mm my 111 me Esuube my my I mm 100 Smxhze r112 macuhung mp ma plate a me his afyaux unsm burner amma my mm Yau ma yam bench pmm HERCISE 21L Inoculte Nutrient Agquot sum from slum cuhun mm r112 snaps m altexuse 2A ma matuhze a mmuummxmxsmm minutmxwm amansme me pmpexmy m tnnsfex maculum m a Shmsux t EXERCISE 2C human Nam A Slnl mm p11 culn e snap 11m may step 2 cooun n mum may Whue haldmgyaur mum matuhung inapwnh mum e m rheMzL39mL39DL39L39MmleMphmtmmxe Can r112 macuhnng map by my muthmg 1 m m are at me am 5mm what 15 m suppanmg my gmwm lynu shauld hear me p mu m i 1 Dr 2 secandsl 39 7 sun s man u cum comquot Wm r112 mus manaap much r112 mm at ans Bahama whiny erhdnw r112 map ma xepuce me m m me plate Culture sun 4 Manila 10 um lamquot mm sunn campme r112 altexuse as mmnaned Mauve m 2mm m INCUBATION Note It is optima to incubate at 37C and record data in 2448 11 o urs Most of the microorganisms we will be studying grow best at 37C body temperature We will attempt to slow their growth by incubating at a cooler 25 30C room temperature This will be done in order to prevent overgrowth or excessive contamination until the next laboratory period which is in one week Your instructor will advise in the proper grouping and labeling of tubes and their location for incubation RESllLLLTABLEJJ NAME OF BROTH APPEARANCE ON SLANT 0R PLATE ORGANISM DENSITY cummmucx CULTURE COLONY COLONY COLOR SIZE mm TEXTURE ASSIGNED CULTURES P gment SHAPE Micrococcus Iuteus X X Bacillus suth39II39s X X X X STUDENT LULl Uth Bsubt7391139s broth X X B suth39II39s slant X X X X M Iuteus slant X X X X 111 FIGURE 12 EXAMPLES OF EACTERIAL GROWTH PATTERNS A CHARACTIRISTICS or COLUMN SIIAP Crreoxor Roono Irregular colony Puncufurm colony regardless may Spread argm zype pmpomr colony Dfm thzoro or rumrhke colony wuh elongated bronehmg I CHARACTERISTICS 01 comm IDGIS Emu Asummh Ummau wavy mm mooomo croummmoma Scallnyed FIGURE 12 CONTINUED C CHARACTERISTICS OF COLONIAL ELEVATION FLAT RAISED CONVEX u A RAISED MARG INS UMBONATE CRATERIFORM HEAPED CONCAVE D CHARACTERISTICS OF GROWTH IN BROTH A Pellicle on the surface B Sediment on the bottom C D Uniform turbidity Control 113 W NUTRIENT BROTH NUTRIENT AGAR SLANT NUTRIENT AGAR PLATE COLONY CULTURE H How would you determine if your culture media were sterile N What are the signs of growth in a liquid medium LN Why do we disinfect the bench top before and after our laboratory period Why are culture tubes held somewhat horizontally when transferring cells rather than vertical U What is a pure culture 114 F 1 9 9 What is colonial morphology Why should long hair be controlled in a microbiology laboratory How dose this apply to a patient care situation Why do you ame the neck of the culture tube when it is opened Name two types of solutions that may be administered to a patient by intravenous injection and therefore must be sterile How would your know if it were sterile 115
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