Lab Report ASAP (2 PAGES)

· Determine the maximum growth rate

· Develop a relationship between cell counts and absorbance

· Bacteria grow at different rates depending on the strain and the temperatures.

· 50 ml LB in 250 ml flasks – 2

· Sterile distilled water for dilutions – 3, 100ml bottles

· Sterile microfuge tubes

· Sterile serological pipettes – 5ml

· Sterile Falcon tubes

· LB Plates – made in previous lab

· Alcohol

· Glass spreaders

· Sterile 1000 ul tips

· Sterile 200 ul tips

· Sterile test tubes

· Distilled water

· Spectrophotometer

· Cuvettes

Notes on preparation –

· Two days before the lab inoculate E.coli into 10 ml nutrient broth tube and incubate at 37oC

· The night before inoculate a 50ml/250 ml flask with 1 ml of the broth from the culture. Shake overnight at 37oC

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· Aseptically take a 2 ml sample after inoculation = T0

· Place in shaker set at 37oC and shake at 300 rpm

· Take 2 ml samples using a 5 ml pipette and place it in a sterile Falcon tube at:

· 20 min

· 40 min

· 60 min

· 80 min

· 100 min

· 120 min

· 150 min

· 180 min

· Note you may need to gather some samples outside of normal class times

· For each sample you will need to

· dilute it and plate on LB plates

· measure absorbance at 600 nm

· Absorbance

· Take a 1 ml sterile sample and place it in a cuvette. Measure the absorbance at 600nm. If needed, dilute the sample with DI water so it is in the 0.05 to 0.7 range. Record the absorbance and the dilution. Multiply the absorbance value by the dilution to get the actual value.

· Use the absorbance at 600nm to estimate the number of cells using the website http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp calculate the amount of number of cells present in your sample.

· Dilution and plating

· Use aseptic practices

· For each samples, set up sterile Eppendorf tubes containing 1000ul of sterile DI water .

· Remove 100 ul from each tube using a sterile pipette tip.

· Take 100 ul from the sample you took from the flask and add to the first dilution tube: this is the 10 -1 dilution or 1/10th ). Mix the 10-1 dilution in the vortex mixer for 20 seconds, then take a 100ul sample and place it in the next tube. Continue until you have a 10-7 dilution

· Sample

· 10-1

· 10-2

· 10-3

· 10-4

· 10-5

· 10-6

· 10-7

· Using an estimation of the cell counts from absorbance select the dilution that will give you between 30 to 300 colony-forming units (cfu). Remember you will be plating 0.1 and not 1ml so take this into account with your dilutions. Also plate the next highest and next lowest dilutions.

· Identify the appropriate dilution of the sample in sterile water in sterile tubes so one of the plates has between 30 – 300 cfu.

· Plate 100ul ml of suspension on the plate in duplicate

· Dip a glass spreader in alcohol and flame (Note: do not heat in the flame) . Use the spreader the inoculum on the plate.

· Incubate plates upside down at 37oC for 24 hours. Store plates at 4oC until needed

· | Time | absorbance (600nm)) | dilution | Final Absorbance |Aligent CFU|

Cell counts

· |Time | Dilution | CFU plate 1 | plate 2| plate 3 | Average cell count (cfu/ml) |

Calculate

· Estimate the maximum doubling time using the growth curves from Aligent and also your CFU data .

· See video

· https://www.youtube.com/watch?v=W6OkbTAuQVc

· For more information, see details under assignments.