Water Tube 4

(Biology Dept.). 0.1 ml of E.coli K or 0.1ml of E.coli B was added to the 10 fold dilution. Using soft agar technique, the growth media mixture with E.coli was plated and incubated.

14. Use the same loop and technique to spread the same cell suspension (+) on the LB+AMP agar plates. Dispose of the sterile loop in a beaker of germicide.

# of plaques/(volume plated x reciprocal of the dilution factor of the dilution tube used)

In part II of the lab six small glass tubes were obtained in a test tube rack. Ten drops of distilled water were then added to test tube 1, five drops to tubes 2-4, and no drops in tubes 5 and 6. Five drops of 0.1M HCl were added to test tube 5 and five drops of 0.1M NaOH to test tube 6. Five drops of enzyme were then added to all tubes except tube 1. Tube 3 was then placed in the ice bucket and tube 4 was placed in the hot bucket at 80-900C for five minutes, the remaining tubes were left in the test tube rack. After the five minutes five drops of 1% starch was added to every tube and left to sit for ten minutes. After ten minutes five drops of DNSA were then added to all the tubes. All the tubes were then taken and placed in the

The 20ml beaker was washed with dichloromethane to collect the solid left in the beaker. The filtered solution was collected in the 25ml filter flask. Then, the filtered solution was transferred to a clean centrifuge tube and the solid compound was transferred to a clean test

In the 10-3 pasteurized sample, the plate exhibited 71,000 cells/mL. The results of the additional dilution samples contained too few colony forming units to count. However, in the 10-7 dilution, although the plate demonstrated 12 colonies, there should have been no colony forming units on this plate. The reasons for this could have been that this sample was contaminated from “double-dipping” the sample before dispensing it onto the plate or when using the pipette, it mistakenly was inserted in a higher concentration sample and then immediately to a lower concentration sample before it was dispensed onto the plate.

Preparing serial dilutions of the alfalfa water will ensure an accurate count. The plates are incubated until you see visible colonies, usually 18-24 hours. The colonies you see growing on the plate are considered to have started from one viable bacterial unit but because bacteria are usually not found as individuals, the colony you see may have started from a single cell or a group of cells. The results are reported as colony forming units (CFU’s).

The plate count or viable count is a method that is used to count the number of bacteria in a given sample, it depends on diluting the sample serially by adding 1 ml of the stock culture to 9 ml saline, and then adding 1 ml from the previous tube to another 9ml of saline, then it is planted on an agar to get isolated colonies of bacteria; the number of colonies is used as a measure of the number of living cells in the sample. Some kinds of bacteria form multiple cell arrangements such as chains, so the bacterial units on the plate are referred as colony forming units; because they might be clumped together. A countable plate contains 30-300 colony if the this range is exceeded then the plate is considered too numerous to count, and if the number

Aseptically we transferred 2 drops E. Coli B broth culture to each of the 5 soft agar overlay tubes.

The micropipette is used to collect very small amounts of liquids that an average pipette would not be able to accurately collect. For this lab, one needs to gather two tubes which will each contain 250µL of solution. One tube will then be filled with the +pGLO plasmid and the other will contain -pGLO. The pGLO plasmid is NOT transmitted into the -pGLO tube. The solution the pGLO plasmid is in is called transformation solution and is used to soften the membrane of the E. coli cells with the calcium chloride it contains. The tubes must remain in an ice bath for ten minutes to bring their temperature down so that they will react strongly when exposed to the heat shock. Whenever researchers are transferring the E. coli bacteria from tube to tube, they must wear protective gloves and sterile loops provided to them. Researchers need to obtain and label four plates: +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, and -pGLO LB. Amp stands for ampicillin and ara stands for arabinose. Their plates contain a base gelatin of Luria-Bertani broth. Then, once the tubes are very cold on ice, researchers place the tubes in 42-degree celsius water for fifty seconds exactly in order to activate the heat shock of the E. coli cells, prompting their membranes to become more permeable and susceptible to accepting the pGLO plasmid. After the

Plates were incubated for 18 hours, then refrigerated as necessary until they could be scored. All plates developed a halo – a small visible ring around the dot, probably caused by the spread of the treatment across the surface of the agar after application. Some plates developed an inhibition zone – an often larger ring around the dot with less growth than on the rest of the plate, presumably caused by the inability of many bacteria to grow where a treatment had diffused through the agar. The boundaries of the halo were generally well defined, while the boundaries of an inhibition zone varied in definition. For each plate, three measurements were taken of the halo and, where applicable, the inhibition zone; the mean of these three measurements is shown in Table 1. After scoring, a sample of bacteria was swabbed from inside the inhibition zone (or, where there was no inhibition zone as in the controls, from a ring around the dot) and allowed to grow in liquid medium for 24 hours. This procedure was repeated until plates 4-6 all developed complete resistance to triclosan, as evinced by the loss of an inhibition zone. In the fourth generation, samples from plates 1-3 were plated on triclosan to provide an additional control against the possibility that inhibition zone loss was a consequence

4. After 5 minutes add 1ml of Benedict’s solution to each test tube. Place the 3 tubes at the same time in a boiling water for 5 minutes.

100 µL of bacteria and fungus from freshly prepared culture was taken in the pipette and poured in the middle of the respective petri plate. Remove excess inoculum by lightly pressing the swab against the tube wall at a level above that of the liquid.Using a cotton swab that has already put in UV light, the bacteria and fungus was spread evenly on the surface of the plate so that bacteria and fungus were spread in each corner of the plate and dried for 4-5 minutes Inoculate the agar by streaking with the swab containing the inoculum. Rotate the plate by 60° and repeat the rubbing procedure. Repeat two times. This will ensure an even distribution of the inoculum. Allow the surface of the medium to dry for

Usually, to obtain useful plates with most samples, the loop-dilution procedure will be conducted when doing pour plate method. This procedure is based on a roughly quantitative dilution of the original sample in agar medium.

A sterile pipette was used to add 0.1ml of E. coli culture to the pH 3.0 tube. This was then repeated for the tubes at pH 7.0 and pH 9.The tubes were then incubated at 37oC for 48 hours. This was then repeated for saline culture of Saccharomyces cerevisiae but incubated for 72 hours at 25oC.