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Experiment 1: Direct Counts Following Serial Dilution Germicides-(Answered)

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Please help me fill in the table in the attached file below. Thank you


Experiment 1: Direct Counts Following Serial Dilution

 

Germicides are a type of chemical means to reduce the number of microorganisms, not totally

 

eliminate them, on a surface, in a liquid, or on a person. Disinfectants are germicides designed for

 

use on inanimate objects (floors, countertops, instruments, etc.) while antiseptics are designated for

 

use on living tissue. In this experiment, you will soak filter paper disks with various chemicals then

 

place the disks on a confluent lawn of bacteria and look for a zone of inhibition (clear area)

 

surrounding the disk. The size of the zone of inhibition is proportional to the growth inhibiting

 

properties of the chemical.

 


 

Materials:

 

60 mL Sterile phosphate buffered saline

 

(PBS)

 

6 Sterile snap-cap tubes

 

250 mL Beaker

 

Hot pad

 

10 Disposable sterile transfer pipette

 

8 Disposable sterile plate spreader

 

(6) 5 cm. Petri dishes

 

Nutrient agar

 

Parafilm?

 


 

1 Pair of gloves

 

10 mL Graduated Cylinder

 

Permanent marker

 

*Microwave or other boiling water bath

 

*1 tsp. Soil sample

 

*10% Bleach solution

 

*You must provide

 


 

Procedure

 

Prepare Agar Plates:

 

1. Loosen or remove the cap on the Nutrient agar bottle.

 

2. Place the bottle in the microwave (if you do not have a microwave, place the bottle in a heatsafe bowl and pour boiling water around the bottle) and heat until the entire agar bottle is

 

liquefied. You will need to remove the bottle and swirl every 10 seconds to distribute the

 

heat.

 

Note: If you notice the agar boiling over, STOP the microwave and let the bottle cool down

 

before handling. Hot agar can violently explode out of the bottle if heated too quickly and/or

 

shaken. After boiling has stopped, use a hot pad protecting your hands and remove the

 

bottle from the microwave. Use caution when removing the bottle from the microwave as it

 

will be HOT!

 

3. Gently swirl the bottle to mix the solution.

 


 

4. Slowly pour the liquefied agar solution into the bottom half of 6 petri dishes so that it covers

 

the entire bottom of the dish. It is important that the entire bottom is coated and that the

 

agar is given time to spread out over the dish.

 

5. Place the lids onto the dishes and allow the agar to gel undisturbed. Note: If you will not be

 

using the dishes immediately, store them upside down in the refrigerator after they have fully

 

gelled. Remove from the refrigerator and allow them to sit at room temperature for at least

 

one hour prior to use.

 

Prepare Agar Plates:

 

6. Use your 10 mL graduated cylinder to dispense 10 mL of PBS in a 15 mL conical tube; label

 

this tube ?Stock Soil Solution?.

 

7. Add 9 mL of PBS into each of the other 5 tubes. Label the tubes as follows: 10 -1, 10-2, 10-3, 104

 

, 10-5.

 

8. Label the 6 petri plates as 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6.

 

9. Add the soil sample to the Stock Soil Solution tube. Invert the tube 10 times to mix well.

 

Prepare a Serial Dilution of the Stock Soil Solution as follows:

 

10. Use a sterile disposable transfer pipette to transfer 1 mL of the soil/saline mixture to the

 

conical tube labeled 10-1 . Invert the tube 10 times to mix well.

 

11. Using the same transfer pipette, transfer 1 mL of the solution from the 10 -1 tube to the 10-2

 

tube; invert 10 times to mix well.

 

12. Transfer 1 mL of the solution from the 10-2 tube to the 10-3 tube; invert 10 times to mix well.

 

13. Transfer 1 mL of the solution from the 10-3 tube to the 10-4 tube; invert 10 times to mix well.

 

14. Transfer 1 mL of the solution from the 10-4 tube to the 10-5 tube; invert 10 times to mix well.

 

15. Using a new, sterile transfer pipette for each tube, transfer approximately 0.1 mL ( or 4 drops)

 

from the Stock Soil Solution tube to the plate labeled 10-1. Notice that you are performing a

 

10-fold dilution of the original sample by only adding 0.1 mL, rather than 1 mL. Spread the

 

diluted soil solution evenly over the plate with a separate sterile spreader. Cover the plate

 

with the lid.

 

16. Repeat Step 15 for the 10-1 tube, putting 0.1 mL of that solution on the 10-2 plate. Spread the

 

diluted soil solution evenly over the plate with a separate sterile spreader. Cover the plate

 

with the lid. Repeat this process for all the tubes and plates, remembering to use clean

 

pipette for each tube and to plate the solution on the next lower dilution plate (i.e., 10 -2 tube

 

solution on 10-3 plate, etc.).

 

17. Allow plates to air dry, then seal with Parafilm?. Incubate in a warm location (not to exceed

 

37.7 ?C or 100 ?F) for 2 - 3 days (until well defined colonies appear).

 


 

18. Observe the plates and assess which plates should be classified as TNTC or TFTC, and which

 

one(s) are countable plate(s). Count the colonies on the countable plate(s) and determine

 

the population density using the following equation:

 

Pop. Density = CFU/(dilution x volume plated).

 

Record your results in Table 1.

 

19. Pour approximately 5 mL of the 10% bleach solution onto each surface of the remaining agar

 

plates, allowing it to cover the entire surface. Incubate them for 20 minutes, and then pour

 

the bleach down the sink with running water.

 

20. Seal the petri dishes with Parafilm? and dispose of them in the trash.

 

Table 1: Experiment 1 Growth Results

 

Plate

 

Classification

 

CFU/plate

 


 

10-1

 


 

10-2

 


 

10-3

 


 

10-4

 


 

10-5

 


 

10-6

 


 

 

Paper#9210300 | Written in 27-Jul-2016

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