I expect to see bacteria growing on or near the streaked line on the LB agar and that the area where the thumb was pressed on the LB agar before washing would be more dense with bacteria than after washing of our hands with soap and drying them. As for the Gram’s staining, I expect to see the bacteria with different colours (e.g. blue, purple, red) revealing the thickness of their cell walls. The shape and arrangements of the bacteria would be visible under the microscope.
Procedures
These were the procedures used.
Preparation of LB agar plate
∙Melt Luria Bertani Agar (LB) in microwave oven.
∙Draw lines at the bottom of the dish to divide dish into 1 half and 2 quarters. ∙Label the to quarters as ‘before’ and ‘after’.
∙Opening the petri dish at 45 degrees, pour melted LB agar into petri dish to fill ¾ of the base
∙Swirl the petri dish gently so that the LB agar cover the base evenly.
∙Leave it to solidify.
Culturing of bacteria
(A) Culturing bacteria from collected sample
∙Dip the inoculation loop into the liquid sample.
∙Opening the petri dish at 45 degrees, streak the sample onto the LB agar. (Use half the dish)
∙Close the petri dish immediately
(B) Test if washing hands reduce bacteria on hands
∙Press thumb gently on the part labeled before.
∙Close the petri dish immediately
∙Wash thumb with soap and wipe dry
∙Press thumb gently on the part labeled after.
∙Close the petri dish immediately.
Gram’s staining
Preparing of the bacteria smear for gram staining
∙Use a marker to draw a circle in the middle of the clean glass slide
∙Drip a drop of water on the marked area.
∙Using the inoculation loop, aseptically transfer a tiny dot of culture to the clean glass slide (area within the circle).
∙Fix and dry the smear by passing the slide very quickly over a Bunsen flame several times.
Procedures (cont’d)
Staining the bacteria
∙Flood the smear with Crystal Violet solution for one minute. Then rinse smear with water
∙ Flood the smear with Iodine solution for thirty seconds. Then rinse smear with water
∙ Flood the smear with 95% ethanol for about two seconds until the violet coloration on the smear disappears. Wash with water again.
∙Flood the smear with Safranin solution for one minute and rinse with water.
∙Blot dry the sample with a paper towel.
Microscopy
∙Rotate the nose of the objective lens until the 4× objective lens clicks in place.
∙Place the glass slid on the stage of the microscope and secure it with the stage clip.
∙Switch on the power of the microscope
∙Adjust the intensity of the light until it is comfortable to the eye.
∙Under the 4× objective lens, rotate the coarse adjustment knob until clear image is obtained. Adjust the fine adjustment knob for a sharper view.
∙ Rotate the nose of the objective lens to the 10× and 40× objective lens for a larger magnification.
∙Shift the glass slid towards a less dense area if the image is too crowded and cannot be seen.
Procedures (cont’d)
Precautions-Before doing anything, remember to put on lab coat and gloves and surface sterilize. Always open the petri dish at about 45 degrees, never talk while doing this and close the dish immediately after that to prevent contaminations.
Preparation of LB agar plate-Firstly, we divided the petri dish into 3 main section, a half and two quarters. Next we label one of the quarters as ‘before’ and the other as ‘after’. Thereafter we opened the dish at forty-five degrees and poured in the melted LB agar into the petri dish until the dish was ¾ filled. We covered the dish immediately and swirled it gently until the LB agar covered the base completely. Finally, we left it on the bench to solidify.
Culturing of bacteria
(A) Culturing bacteria from collected sample-The inoculation loop was dipped into the liquid sample (yitagen, yakult, fish tank water, left over water) which was collected. Carefully, we used the inoculation loop to streak the sample onto the LB agar (the biggest section).
(B) Test if washing hands reduce bacteria on hands-Gently press thumb on LB agar on the area marked before. Thereafter we washed our hands with soap and wiped it dry. Once again, we pressed our thumbs on the LB agar on the section marked after.
The petri dish was thereafter incubated at 37degrees Celsius for about a day, to let the bacteria grow.
Gram’s staining
Preparing of the bacteria smear for gram staining-After using a marker to draw out a circle on the glass slide, we flip over the slide and dripped a drop of water on it. Thereafter, using an inoculation loop, we aseptically transferred a tiny colony (petri dish 3, before section) to the glass slide and smear it within the marked circle. Thereafter, the glass slide passed through a Bunsen burner flame very quickly. This would kill the bacteria so that the staining would be successful.
Staining bacteria- Firstly, we flooded the smear with Crystal violet solution for 1 minute. Next the glass slide was washed with water. Again, we flooded the smear with iodine solution for 30 seconds and washed with water. Thereafter, we flood the smear with ethanol (for about 3 seconds), until the violet coloration on the smear disappears. Finally, the smear is flooded with Safranin for 1 minute, rinsed off with water and blot dry with a paper towel.
Microscopy-After the nose of the objective lens is rotated to the 4x objective lens and the glass slid is placed on the stage and secured by the stage clips, the stage is lifted up to the highest with the coarse adjustment knob. The power was then switched on and the light intensity is adjusted until comfortable to the eye. The course adjustment knob is adjusted again to obtain clearer image and the fine adjustment knob is adjusted to get a sharper image. The objective lens was rotated to10x and 40x objective lens to view the bacteria at a bigger magnification.
Results
These were the results for the culturing of bacteria.
Petri Dish 1
Sample- Water from fish tank.
Observations
There were many colonies of bacteria growing on the line where the sample was streaked. The colonies were about 0.5 in diameter. There were also several confluent growths.
As for the bacteria which was collected from the thumb, there were many small colonies and a big confluent growth in the center in the before section. There were also a few colonies of bacteria in the after section.
Results (cont’d)
Petri dish 2
Sample- left over water
Observations
There were many small colonies growing along the line where the sample was streaked. They were very small and most where just a little bigger than a full stop. There was also a confluent growth that was about 1.5 cm in length.
As for the bacteria which was collected from the thumb, there were many colonies and a few big confluent growths in the before section. There were a couple of colonies in the after section.
Results (cont’d)
Petri dish 3
Sample- Vitagen
Observation
There was only one very small colony in the sample section, which was not growing on the streaked line.
As for the bacteria which was collected from the thumb, there were many small colonies of bacteria in the before section. There were uncountable numbers of colonies of bacteria in the section after. There were also a few confluent growths growing in the center.
Results (cont’d)
Petri dish 4
Sample- Yakult
Observations
There were only two very small colonies in the sample section, which were not growing on the streaked line.
As for the bacteria, which were collected from the thumb, there were many small colonies of bacteria and two confluent growths growing in the section before. There were uncountable numbers of colonies of bacteria in the section after. There was a huge confluent growth in the center about 2 cm in length. There were also a few confluent growths growing around the huge confluent growth.
Results (cont’d)
This is a section of the results from the microscopy (Gram’s staining).
Source of sample: Petri dish 3 (before section)
Total Magnification: x400
Observations
The bacteria were all coccus shape. Most of them were growing in chains, but there were some bacteria growing in huge clusters. Their colours after Gram’s staining ranged from dark purple to a reddish brown colour.
Discussion
There were some positive as well as negative results in the experiment done. They were discussed and here are some of the reasons to what happened.
Petri Dish 1 (sample-fish tank water)
There were many small colonies of bacteria growing on the streaked line on the LB agar. This may be caused by waste produced by the fish, the left over food of the fish and some other conditions. Our hypothesis for this sample is accepted as the bacteria from the collected sample grew on and near the streaked line on the LB agar.
As for the results of the test for bacteria on the thumb, it shows that the thumb was dirty before washing, as there were many colonies of bacteria in the before section. However, after washing with soap and drying, there was much lesser bacteria then before, and the thumb was cleaner. Our hypothesis is accepted in this case, as results shows that washing our hands with soap would kill a large amount of bacteria.
Petri Dish 2 (sample-left over water)
There were many very small colonies and one confluent growth of bacteria growing on the line where the sample was streaked. This shows that left over water is contaminated and contains a lot of bacteria. The bacteria in the water have multiplied itself when left in the bottle. These bacteria probably caused so many colonies to grow on the LB agar. Our hypothesis for this sample is accepted as the bacteria from the collected sample grew on and near the streaked line on the LB agar.
As for the results of the test for bacteria on the thumb, it shows that the thumb was quite dirty before washing, as there were a lot of colonies of bacteria in the before section. However, after washing with soap and drying, there was lesser bacteria then before, and the thumb was cleaner. Our hypothesis is accepted in this case, as results shows that washing our hands with soap would kill a large amount of bacteria.
Petri Dish 3 (sample-vitagen)
In this sample, there was only one colony of bacteria, which was not growing on the streaked line. The reason to this result is due to the vitagen (sour), which is more acidic then LB agar, which is about ph7. Since the LB agar is less acidic than the bacteria, the bacteria in the vitagen (Lacto bacillus) didn’t grow, unlike how it grows in liquid media in the factory. The small colony of bacteria is probably a contamination caused by speaking while doing the experiment. Therefore our hypothesis in this case is rejected.
As for the bacteria which was collected from the thumb, there were many small colonies of bacteria in the before section. However, there were uncountable numbers of colonies and confluent growths of bacteria after washing and drying of the hand. One possible reason for this unusual result is that the cloth that was use to wipe the thumb had a lot of bacteria. Therefore our hypothesis in this case is rejected.
Discussion (cont’d)
Petri Dish 4 (sample-yakult)
In this sample, there were only two yellow colonies of bacteria, which were not growing on the streaked line. The reason to this result is due to the yakult (sour), which is more acidic then LB agar, which is about ph7. Since the LB agar is less acidic than the bacteria, the bacteria in the yakult (Lacto bacillus) didn’t grow, unlike how it grows in liquid media in the factory. The small colony of bacteria is probably a contamination caused by speaking or sneezing while doing the experiment. Therefore our hypothesis in this case is rejected.
As for the bacteria which was collected from the thumb, there were many small colonies of bacteria in the before section. However, there were even more colonies and a few huge confluent growths after washing and drying of the hand. One possible reason for this unusual result is that the cloth that was use to wipe the thumb had a lot of bacteria.
Therefore our hypothesis in this case is rejected.
Microscopy (Gram’s Staining)
After Gram’s staining the bacteria, the colour of the bacteria on the glass slide rangers from pink to dark purple. Their shape was coccus and was in both chains and cluster arrangements. Our hypothesis is accepted in this case, as results shows that the shape and arrangement were visible under the microscope. The colour of the bacteria after Gram’s staining also revealed the thickness of their cell walls.
Problems faced
The biggest problem that was encountered was, contamination, which was extremely visible in Petri Dish 3 and Petri Dish 4 (culturing bacteria from sample). The main reason to the problem was probably due to talking and sneezing while culturing the bacteria. Another problem in Petri Dish 3 and Petri Dish 4 was that the cloth used to wipe the thumbs probably had a lot of bacteria, which made the experiment an unfair one. The third problem is that the bacteria in petri Dish 3 and petri Dish 4 couldn’t grow due to the LB agar being less acidic.
Recommendations
The biggest problem that was encountered was, contamination due to talking and sneezing during the experiment, which was extremely visible in Petri Dish 3 and Petri Dish 4 (culturing bacteria from sample). Another problem in Petri Dish 3 and Petri Dish 4 was that the cloth used to wipe the thumbs probably had a lot of bacteria, which made the experiment an unfair one. The third problem is that the bacteria in petri dish 3 and petri dish 4 couldn’t grow due to the LB agar being less acidic.
One good way, which I recommend to reduce contamination due to talking and sneezing during the experiment, is to use mouth mask that covers the mouth and nose. As for the problem about the dirty cloth used to wipe the thumbs, I recommend that clean paper towels should be used to wipe the thumbs dry so that the experiment is a fair one. Lastly, for the third problem, I recommend that a sample used should have a ph lever closer to that of LB agar.
The Conclusion
The main aim of the life science lessons is to learn to handle and culture bacteria and observe the bacteria. If the steps of the experiment were carefully followed, I can conclude that bacteria in the collected sample will grow on the streaked area (if there’s no contamination and if the sample has a ph level not to far from LB agar), washing hands with soap can reduce bacteria (if the cloth used to wipe dry is clean) and Gram’s staining would make the shape and arrangement of bacteria visible under the microscope as while as the thickness of their cell wall.
References
Kindersley, D. 1993. The Science Encyclopedia. Pg. 313. Convert Garden Books
Microsoft Encarta Encyclopedia Standard edition 2002.
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