Microbiology Lab: 2, 3, 4, 11

Ch 2,3,4 II CH#2 Brightfield microscopy bnght field dark object false? can be used to obseme unstained microorganisms in wet mounts /hanging droy slides - Brownian movement - the movement movement Hand S trikes object causes it to bounce vibrate at th e sam e time 1 keep their positions actual motil le- actually more from one position to another. movement movement W more directed occassional spin/rolls to microbiomes microbial communities in environments Ex motozoa, algae fung) bactena found in pond water. - looking at Michorganisms is usefalin determining size e,shape and movement wet mount useful fast way to see bactena but hard to see movement Hangingdrop technique better for larger microbes and motility * (petroleum Jelly seal reduces evaroration of the drop of Auid ) better for Wet mount ( the most basic one) STEPS (small/nonmorite) 1 suspend infusion 1 stiming/shaking) 2 use pausteur pipette to transfer a small drop onto a shde 3 Handle coverslip by its edges and Place it on the drop 4 gently press T end of pencil or loop handle (to secure) 5 Place on slide on the microscope and start on lowest power and go to the highest better for Hanging-Drop P recedure (bigger/motile) Dobtain I clean hanging dror (depression) slide 2 Pick up 9 small amount of petroleum jelly on a toothPIck 3 PICK UP a COVEISLIP (bvits edges), and carefully touch the Jelly w/an edg 0 the coverslip to geta smal rim of petroleum Jelly, Repeat T the other 3 edges, keeping the petroleum Jelly on the same side of the coversive 4 place bactera in middle of coverslip 5 place depression slide over the covership I Quickly invert it /drop should be hanging at his 7 view it under memocope

1st solid culture media was usedin 1812 and was made from potatostarch, coagulated egg white and meat aseptictechnique are procedures used to avoid introducing unwanted microbes and to present spreading membes Wherethey are Owanted insulating loop- Michrome wireheld with an insulated hundk flaming Stenliang the inoculating tube before and after are usingle-use plastics loop are plamed-already stenle Held over burner or in electric incenerator inner horton corner'sf blue flame (hottest part) agur comes from seq weed Angelina Hess suggested use of agai for growing bactenal cultures CH#3 Microblome community of muches that inhabit a Particular environment environment microbiomes can exist on lab benches environment objects? human merobiomes on humanbody/Skin Most of time are harmless mediums used to culture bactena require maconutnents (carbon, ntrogen, phosphons and sulfur) t an energy source, and growth factors chemically defined medium a medium whose exact chemical composition IS known is called a chemically defined medium complex media: media for which the exact chemical composition vanes slight ly from batch to batch Nutnent broth- commonly used nigued complex medium Nutnent agar - so lidited nutner both that had agar added to the Agar extract from manne red algae / useful in culture media remain sold dunng microbial growth sugueties a 100'G remains liau until its cooled at (40°C once solid, can be Incubated at 100'6 and remain solld' - media needs sterilization a free preparation most common steam Sterilization 2 autoclaving steam under pressure heated to 121°C it 15 Pounds of pressure X 15 mins

growing bactena large petriplates containsolid media and allow C surface are examination microbes are (noculated 1 introduced to butnett agar(sold)or nutrent brtth (liquid) Incubation period - ba tena grow after broth will become toxield /cloudy 11 growth - bacterial by grinth turbidity inbroth determined agar - you will see bactena with naked eye =growth spellicle onsurface Colony population of cells that onses from a single bacterial cell Hocclent suspended in broth CFOC Colony forming unit) -colony that anss from a group of the bottom sediment at same microbes attached to one another preparing culture media J i prepare 100m of nument broth in 250ml flask 2 add 100ml of distilled water 3 add nutn ent broth powder MIX untilthe powder is dissoived 5 pipette 5ml of broth into each test tubes (3) and cap each 6 Label it Nutnent broth" (2 will be autor laved , last one nont) 7. add agar to 1 remaining 85 ml of nutrent brot 8 Boll until agar 9 to autoclave flask and B premonstubest Label hutment agar 1 allow cooling at room temp or 450 water both pounng plates Petn (dishes I 4 Plates in front of you (unopened) 2 Hold flask at angle MS remove stopper T 4th and 5th finger ofo the hard 4 Heat mouth of Has K 2-3 times 5 remove cover from first dish T same hand thats holding stopper 4 Pour agar QUICKLY /neatly (cover bottom about 5mm) 7 repl ale corer 8 do other petn dishes 9 Shirl all 4 plates to cover any empty spaces 100 000 touch lidandagai) 0 Have slight 4 open 15 min to I condensation als card empty flask in discard area

colonies can appeal whole-colony ap e arance -can be circular, irregular, Biconvex, Filamentous, Rhizad shiny matte, granular (long perjections) I (MAAS) hanino 2 margin (edge)can be (not-11kc) entire undulate punctiform lobate Filamentous Curled 3 elevation raindropshape rare. Flat raised convex umbonate CH 4 Allculture media are stenlied prior to use stehlization IS accomplished using an autoclare Different culture media Broth cultures provide larg#'s of bucteria in a small space and are easily transported Agar slants tubes containing solld culture media that were lett at an angle and solldised L solld growth surface easy to storeftransport leaster than petri plates) -Agar deep- agar solidified at bottom of test tube used for bactena that require (ess oc) - -semisolid agar only 0.5'07% (instead of usuall.5%) IS used to dennine whether bactena IS motile mottle bactern mores away from pointof inoculation, giving appearance of an inverted pinetree.

simple stams -1dyelbasro STAINS differentiation gramstownand acid fast stain flagella Inoculation on the mtent of the procedure youan performance inoculating 100P A Indulating needle. 7 inoculating 100P used for nutnet broth and nutrentagar in oculating needle used for semisolid agai Hold inoculating toor in dominant hand steps broth tube mother hand edge of 2 3 while holding stenle 100P, remove cap of tube Hold it in hand stenlife loop hottest part (inner blue flame) (2-4 secs) 4 Hold to be in front of Sterillier 5 immerse the stenle loup into broth and getculture 6 remove loop 7 resterilize mouth of tube 8 recap tube q repeat always hold culture tubes and uninoculated tubes at about a 20° angle to minimize the amount of dust that could fall into them and dont tip tube too far b/c the liauid will leak out CH dont let top of tube touch anything!! AMA WB Staming bactena enhances the contrast between bacteria and the surrounding matenal X and permits observation of greater detail and resolution than do net -mount procedures simple stains : only reagent IS used and all bacteria are similarly stained (basic dye) thaDey differential stains multiple reagents are used and bacteria react differently to them DOLLARS structural stains used to Identify specific parts of michorganisms gram mixed cultures (more ethan colony /bacteria) harder to study. to study To study growth characteristics pathogenicity metaborism, ATB suceptibility more indepth of other charactensries requires a PURE CULTURE ( contains I microbe) use type of purecultures In 1880 Robetkoch prepared solld media that helped to (Date

isolating bactena Edilotion methods are used I Streak 2 spread plate 3 your plate solid f Streak: loop IS used to Streak mixedsample many times over culture medium in g petriDish teadly causes bactena to fall off 1001 one by one and be streaking distributed repe over the agar surface, where each (eb develops into a colony label bottom of Petn Dish. STEPS 1 2 Flame 100% 3 attain 1001ful of broth culture. 4 I tone edge of petricover, hold in same hand and do 1st streate petri beling can 5 6 Flame loor tum Plate - Strea Lasan it 7 Flame \ loop etc etc. areas that touch are reinocularted (going back aemossaprenous Scenton) 2 Spread Plate technologic - small amount of previously diluted specimen IS spread over the surface of a solid medium using a spreading rool 3 pour Plate technique small amount of diluted sample IS mixed I melted agar and pouted into empty, Sterile Petri Dishes STEPS label bottom of 3pennish a label dilution blanks

anything below 25 Is inausate b/c single contaminant causes at least 4/emr 25-250 colonies have to be 25-250 to be able count (25- 0 accurate 4 Derri 7250 - too many toor and s Fill beaker T hot (45-50%)watter (3-6cmdeer) 4 place3 tubes of melted agar In beaker 6 select mixed b10th culture red agar is beef extract yeast extract 1.8% Nac I Distilled water Sheep Blood. Beta lalpha gamma - all cells almost all cell cells usually blood thayer -martin has 4 antibiotics Vancomyan Selective for gononhaea COLISHK Manill has gononman Nystatin Trimethoprim EMB- selective for gram(-) bactena Lowenslein -Jenson medium MYCObactenum (1B) Contain PNC Stains green contains malachite green to inhibit growth of gram(+) and gram() bactena limits growth to mycobactena species only.

Differential media Special media Selectivemedia macconkey agar ennched media thayer martin medium OLEDIOGAY EX: Blood agar eosin methylene blue TCBS agar chocolate agar campylobacter agar lowenstein - Jenson method. XIDagar 14 n Anaerobic media Transport media obugate aerobes Facultative anderobes Aeroto errent angerobes