Instruments and Materials
- Bacterial suspension (E. coli) in a small test tube (1:10 diluted overnight culture).
- Plastic droppers, 8 pipettes/group
- Petri dishes, plastic, sterile, > 16 plates/group
- Spreader, plastic, 8/group
- Micropipette tips (tips for P200 and P20)(to be autoclaved)
- Flasks
- Normal saline(to be prepared)
- Heart Infusion broth: 50 ml.
- Nutrient agar medium plates (to be prepared from powder medium)
- Microcentrifuge tubes(1.5-ml, to be autoclaved)
- Small glass test tubes with plastic caps (to be autoclaved)
- Spectrophotometers
- A sheat of semi-logarithmic graph paper
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Procedures
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Day 1
Prepare followings:
- Sterile saline
Prepare 100 ml of normal saline (0.9% NaCl) in a 200-ml flask. Cover the flask with double layered aluminum foil and autoclave. Keep on the laboratory bench.
- Heart Infusion Broth
50 ml in a 200-ml flask, Stick with a plug and autoclave.
- Nutrient Agar plates: 300 ml agar medium in a 500 ml EM-flask or in a 500-ml medium bottle. Two flasks or bottles.
For 16 plates. Write "periods and dilutions" (for example, "0 hr, 10-5") on the plates.
- Small sterile test tubes
13 x 100 mm, glass, with plastic caps. 6 tubes, Autoclave and dry at 37 C overnight (for measurement of optical density).
- Micropipette tips
Put micropipette tips into cases ( Ware plastic gloves to handle the tips, 100 tubes/lack for P200, 50 tubes/lack for P1000) . Cover with aluminum foil and autoclave. Dry at 37 C overnight.
- Microcentrifuge tubes (sterile, microtubes)
Put 40 tubes into a paper bag; autoclave; dry at 37 C overnight.
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Day 2
- Dispense sterile saline into sterile microcentrifuge tubes each with 0.9 ml using micropipette (Pipetman P-1000) (5-7 tubes x 4-5 times = 20-35 tubes). Write "periods and dilutions" (for example, "2nd, 10-5" or "4:25, 10-5" or "11:10am, 10-5").
- Transfer a 2-ml portion of Heart Infusion Broth (HIB) into sterile small tube for "blank" for O.D. meter.
- Inoculate a drop of the cell suspension of the E. coli culture into 50 ml of HIB prewarmed at 37 C. Mix the inoculated culture thoroughly.
- Immediately after mixed, transfer ca. 2-ml portion of the inoculated culture into a sterile small tube. Immediately, start incubation of the culture at 37 C incubation room with shaking.
- Determine optical density of the culture at 600 nm using spectrophotometer (using Bausch & Lamb Spectronic 20 or alternatives such as Shimadzu).
- Dilution and inoculation for cfu culture
- Transfer a 0.1 ml-portion of the obtained culture into a microtube containing 0.9 ml salineusing Pipetman; close the cap and mix thoroughly (Dilution:10-1).
- Repeat the previous step to dilution of 10-7.
- Inoculate a 0.1 ml-portion of each dilution onto a nutrient agar plate (from high to lower dilution) using Pipetman.
- Spread inoculum on agar plates with spreaders.
- Dry the surface of the agar plates.
- Incubate overnight at 37 C.
- Repeat dilutions and inoculations as previous step at 2, 4, and 6 hr (You may incubate up to 8 hr when you want to incubate and check cfu and OD more.)
Day 3
- Cout the numbers of emerged colonies on agar plates which have 30-500 colonies/plate by dotting the colonies on the surface of the Petri dishes with a felt pen.
- Select the number of colonies which are suitable for estimation (i.e., 30-500 colonies per plate).
- Record the dilutions used for estimation of the colonies.
- Caluculate the mean numbers if the you use the plates of two dilutions for estimation of the colonies (e.i., when you had 35 colonies at dilution 10-4 and 400 colonies at dilution 10-3, you can use (35X10+400)/2=(350+400)/2=750/2=375 colonies at dilution 10-3).
- Estimate the numbers of the viable bacterial cell in the culture medium.
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Estimation of Viable Bacterial Cells
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Period
(a) | No. of colonies emerged | cfu(viable cell/ml)
| OD 600 nm
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Dilution
| Difinite Number
of colonies [n]
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| [n]×10×1/[dil.]
(b)
| Common logarithms,
(log10)*,(c)
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| 10-1
| 10-2
| 10-3
| 10-4
| 10-5
| 10-6
| 10-7
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| Time 0 ( : )
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| 1st, 1-3 hr. ( : )
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| 2nd, 3-5 hr.( : )
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| 3rd 5-7 hr.( : )
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| 4th 7-9 hr.( : )
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| | Diluted and inoculated | | Diluted, not inoculated | | not diluted, not inoculated
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- * ; Use Microsoft Excel (or OpenOffice Calc) to calculate logarithms if necessary.
Growth curve
Draw the growth curves of the cfu and the optical density on sheats of :
squared graph paper;
X-axis, a; Y-axis, c | 
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| semi-logarithmic graph paper;
X-axis, a; Y-axis, b | 
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(Plot the cfu on a logarithmic scale.)