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Experiment Instrucs
| Question | Answer |
|---|---|
| Preparation of a Culture Medium | Prepare 100 mL of medium by mixing 3 g TSB (30 g/L) and 1.5 g agar (1.5%) in 100 mL distilled water, microwaving until the agar dissolves, then cooling to 60°C. Dispense 7 mL into test tubes for slants and autoclave at 121°C, 15 psi, for 15 minutes. |
| Slant to Slant Transfer | Heat needle, remove cap (without putting it down), remove a small amount of surface growth from slant (no gouging), streak this inside the fresh agar slant from bottom up, replace cap and heat needle. |
| QSP Method | Divide plate, Use a sterile loop to place mixed culture in quadrant 1 and streak back and forth heavily, then streak from quadrant 1 into quadrant 2 with fewer strokes, repeat the process, incubate at 34 C. Flame between quadrants. |
| Pour Plates (Serial Dilution) | Label the bottom of the dish and then pour the agar inside. If you have multiple plates, divide the agar amongst them. Let the agar harden for 5-8, then add diluted cultures, swirl in a circular motion. Put the plates in the bin agar side up. |
| Serial Dilution | Fill up some tubes with buffer (9.9 mL for 10^2 and 9 mL for 10^1). The first tube should have culture added to it as well (0.1 mL). Vortex. Then take 1 mL from the 10^-2 dilution and put it in the next tube. Total ODF of 10^-3. |
| Swab Inoculation (Kirby-Bauer) - Chemical | Swab inoculate surface of the petri plate with broth culture by swabbing half, rotating 90 degrees, swapping again (up to 4 times). Then, using forceps, dip filter disks into subject of interest, then put in quadrants. Incubate and measure zones. |
| RODAC Plates - Chemical | Place one unwashed hand on a RODAC plate, one washed hand on a RODAC plate, and swab a third with tap water as a control. |
| UV Radiation Experiment | Divide a plate into 10s, 20s, 30s, 1m, 2m intervals. Turn on the UV light and use an index card to cover the respective sections as the light passes through them and time passes. |
| Selection of Fungi | Create pour plates of TSA and Mycophil (just pour both of the substances inside). Take them outside and expose them for five minutes. Afterwards, cover them up again and place the plates agar side up. |
| Preparation of a Wet Mount - Broth Culture | Flame loop, place inside culture, and place a drop of the culture on the slant. Cover with the coverslip. |
| Preparation of a Wet Mount - Bacterial Slant | Flame loop, use the loop to place two drops of H2O each about 1 cm from the center. Use needle to touch culture, then mix into first drop of water. Flame needle, then mix that first drop into the second via a line. Cover with the coverslip. |
| Gram Stain | Flood with crystal violet, retain for 1 min, wash & drain excess water by tapping side with towel, flood with iodine, retain for 1 min, wash off then BLOT DRY, flood with decolorizer until purple is gone, wash, counterstain for 2 min, wash and blot dry. |
| Negative Stain | Place a single drop of nigrosin close to the slide edge using loop, flame loop, mix culture into the nigrosin, take another slide and smear the nigrosin mixture all across the first slide. Wait and dry. |
| Catalase Enzymatic Test (Gram Positive) | Use a needle to put your positive and negative cultures on the slide, then add a dropful of hydrogen peroxide and see which has bubbles. |
| Smear Preparation (Gram Stain) | Draw three wax circles (+, -, and UNK), then flame a loop and add a loopful of water to each circle. Add culture to each loop afterwards by mixing it into water. Heat fix by passing through the flame in a figure 8. Water totally evporate |
| Oxidase Test Enzymatic Test (Gram Negative) | Get a small amount of the culture on a swab, put 2-3 drops of the reagent on the swab, wait for a color change in the next 20-30 seconds. |
| Citrate Slant (-) | Using a needle, streak agar slant with your culture then incubate. |
| MR-VP Test (-) | Inoculate slant with MR-VP broth, then introduce culture to the broth with needle. Incubate. After incubation, add 10-15 drops of each reagent for VP. 3-4 for MR. |
| SIM Test (-) | Inoculate SIM medium with your culture using needle. Incubate. |
| Nitrate Broth (-) | Inoculate broth with appropriate culture. Use your needle to introduce a small amount of growth from the slant into the liquid medium. After a week, add 5 drops of each reagent if there's no bubbles. If nothing happens still, add a small amount of zinc po |
| Phenol Red Glucose (+,-) | Inoculate broth with unknown culture. Use needle to introduce growth. |
| Phenol Red Lactose (+,-) | Inoculate broth with unknown culture. Use needle to introduce growth. |
| 6.5% NaCl TSB (+) | Inoculate the bacterium into TSB containing 6.5% NaCl, incubate, and observe for turbidity, which indicates growth and salt tolerance. |
| Bile Esculin (+) | Using a needle, inoculate a slant with unknown culture. Streak several times across the surface. |
| EMB (-) | Streak the bacterium onto EMB agar plate, incubate, and observe colony color and sheen to determine lactose fermentation and growth. Basically QSP. |
| Novobiocin Sensitivity Test | Spread a bacterial lawn on Mueller-Hinton agar, place a novobiocin disk, incubate, and measure the zone of inhibition to determine susceptibility. |
| Mannitol Salt Agar (+) | Streak the bacterium onto MSA plate, incubate, and observe growth and color change to determine salt tolerance and mannitol fermentation. |
| Bacterial Plasmid Transformation | Treat cells with cold CaCl₂ to make them competent, add plasmid DNA, incubate on ice 30 min, heat shock at 42°C for 90 sec, add LB broth, incubate 40 min at 35°C, then plate on selective media. Successful transformants grow on ampicillin plates. |
| Bacterial Plasmid Isolation | Harvest cells by centrifugation, lyse cells with alkaline solution to release DNA, then use alcohol precipitation to separate small plasmid DNA from larger chromosomal DNA and other cellular debris. |
| Plasmid digestion | Dissolve isolated plasmid DNA in water, add 10 µl plasmid to tubes containing restriction enzyme digestion mixes (EcoRI, HindIII, or both), incubate at 37°C for 40-45 min, stop reaction with 2 µl gel loading buffer, then load samples on agarose gel. |
| Gel electrophoresis | Prepare 1% agarose gel by dissolving agarose in 1X TAE buffer, cool and add Eth Bro, pour gel in casting tray, load samples (~15 µl) including uncut plasmid and digests, add DNA size markers, run gel with TAE buffer, and visualize bands under UV light. |
Created by:
smurtab