Krasanya Gaur

Shivani Krishna, Imroze Khan

Life in the Neighbourhood Lab
3rd February 2023
Microbial World Diversity II- Report

Introduction:

We conducted three experiments to interact with the various bacteria around us on the campus.

The first experiment was to compare bacteria deposition from surroundings on our agar plates,

when they were left exposed to bacteria for different time periods and in different ways. The

result for this experiment would give us a comparison between deposition rate of bacteria on a

surface v/s in the air, along with a comparison between the amount of bacteria deposited in

different time periods.

In the second experiment we pressed a leaf surface onto an agar plate to compare the amount of

bacteria on a leaf with our other results, and also to observe the difference in bacteria quantity on

the top and bottom surfaces of the leaf.

The third experiment is an interesting way to maneuver the varying thickness of peptidoglycan

membrane on gram positive and gram negative bacteria to create observable slides.

Method and Sampling Strategy:

Experiment 1-

1. Collect 3 agar plates from the lab and decide where you want to conduct your

experiment.
 2. Mark the agar plates for different durations - 5 mins and 15 minutes. The third one will

be marked as an air sample.

3. Place the agar plates on a surface for the stipulated time period without touching the agar.

4. Carefully close the lid of the agar plates when the given time elapses.

5. Gently wave plate 3 in the air for 30 seconds and close the lid without touching the agar.

6. Bring plates back to the lab and place in incubation at 37 degree celsius for 24 hours.

7. Observe the formation of bacteria colonies on the agar plates and compare your findings.

Experiment 2-

1. Collect 1 petri dish with lid from lab and chose a leaf of your choice

2. Carefully keep the petri dish on the leaf and close it without touching the leaf so as to

avoid contact of hand with the leaf. This could contaminate the results.

3. Bring the petri dish back to the lab.

4. Wear gloves and collect 2 agar plates, one stick and tweezers.

5. In a sanitary area, use tweezers to hold the leaf and place it in an open agar plate.

6. Now use the stick to press the leaf such that it comes into contact with the agar from

everywhere.

7. Use tweezers to remove the leaf from the plate, turn the leaf around and place it in the

second agar plate.

8. Now use the stick to press the leaf such that it comes into contact with the agar from

everywhere.

9. Safely close both agar plates and incubate at 37 degree celsius for 24 hours.

10. Observe the microbial growth pattern on both agar plates.

Experiment 3-

1. Take a slide and clean it to degrease.

2. Add 50 ml of both the given bacteria solution to a pipette.

3. Add liquid nutrient broth in the same pipette.

4. Dilute this using PBS.

5. Add a few drops of this on a slide and smear using a coverslip.

6. Fix the bacteria by keeping a slide over the bunsen burner until water evaporates.

7. Add crystal violet dye and let it absorb for 1 minute exactly.

8. Wash with distilled water.

9. Add grams iodine solution to make the dye stick to the peptidoglycan of cell walls of

bacteria and keep for 1 minute.

10. Wash with distilled water.

11. Add the decolorization agent and let it stay for 15 seconds.

12. Wash with distilled water.

13. Add Safranin and let it stay for 1 minute.

14. Observe slide in 40X zoom.

Observations:

Experiment 1 and 2-
Bacteria largest colony smallest colony shape of
colonies No of colonies size size color colony
most are
creamy white,
one type is mostly regular
5 minute plate 29 1.5 cm 2 mm yellow circles
most are
creamy white,
one type is mostly regular
15 min plate 69 3.5 cm 1mm yellow circles
air sample 0- - - -
smaller
colonies are
regular circles
while larger
ones become
leaf up side 17 4cm 3mm creamy white irregular
flower shaped
colonies with
bronchiole type
leaf down side 9 7cm 5mm yellowish white design

5 minute plate
 15 minute plate

15 minute plate

Air sample
 leaf up

leaf down

Experiment 3-
 gram positive

bacteria

gram negative bacteria

Result:

Experiment 1-

● Larger amount of bacteria colonies are seen in the plate exposed to the environment for

longer.

● Air sample showed the lowest amount of bacteria deposition.

● Similar types of bacteria are seen in both plates.

Experiment 2 -

● Wherever the agar has come into contact with the leaf, bacteria colonies are present. This

is why they are seen in a line instead of scattered fashion like other plates.

● The lower surface of the leaf has more bacteria density, and it can be seen that the

bacteria on the lower surface grow in a particular fashion that looks like concentric

flowers.

Experiment 3 -

● Since gram positive bacteria has once taken up the stain, gram negative bacteria is the

only one visible with safranin stain.

Discussion -

1. Larger the colony the more irregular the shape

2. Many colonies are seen interacting with one another on the plate, such as

superimposition, eating up of one colony by another, joining of colonies.

3. Colonies are seen in a line due to the shape of the leaf when placed on agar plate