LAB 6 : MICROBIOLOGY

LAB 6 : MICROBIOLOGY

Objectives:

v Knowledge of how distinguish the gram-positive and gram-negative bacteria properly.

v To develop the laboratory skill in gram staining in proper ways.

v To be more familiar with gram staining procedure.

v To understand how the gram stain reaction affects Gram positive and Gram negative bacteria based on the biochemical and structural differences of their cell walls.

Introduction :

Aseptic means to be free from microorganisms. Aseptic Technique is the procedure that is performed under sterile condition to prevent the growth of other microorganisms on the growth medium such as the Petri dishes containing the nutrient agar or the pure culture. If the growth medium or the pure culture is contaminated with microorganisms from the environment, it will results in confusion and inaccurate data. Hence, it is important to reduce the risks of these microorganisms to come in contact with the experimental materials.

        Aseptic technique also known as a fundamental and important laboratory skill in the field of microbiology. Microbiologists use aseptic technique for a variety of procedures such as transferring cultures, inoculating media, isolation of pure cultures, and for performing microbiological tests.Proper aseptic technique prevents contamination of cultures from foreign bacteria inherent in the environment. For example, airborne microorganisms (including fungi), microbes picked up from the researcher’s body, the lab bench-top or other surfaces, microbes found in dust, as well as microbes found on unsterilized glassware and equipment, may potentially contaminate cultures, thus interfering with the lab results.

        Using proper aseptic technique can greatly minimize or even eliminate the risk of contamination. In addition, aseptic technique is of utmost importance to maintain pure stock cultures while transferring cultures to new media. Aseptic technique is also essential for isolation of a single species of microorganism from a mixed culture to obtain a pure culture. Furthermore, proper aseptic technique prevents microbes used in the laboratory from accidentally being released into the environment and infecting people working in the laboratory. This is especially relevant when pathogens are being handled.

Material and apparatus :

Ø Agar plate

Ø Bunsen burner

Ø S.aureus and E.coli

Ø Loop

Ø Soap

Ø Hand sanitizer

Ø Slide

Ø Crystal violet

Ø Distilled water

Ø Iodine

Ø Ethanol

Ø Safranin

Ø Microscope

Methodology :

PART 1

(A) Streak plate technique

1) The inoculating loop was sterilize in the bunsen burner by putting the loop into the flame until it is red hot. Allow it to cool.

2) An an isolated colony from the agar plate culture of E. coli was picked and spread each of them over the first quadrant on separate agar plate.

3) The agar plate was covered with the lid and flame the loop.

4) The plate was turned and lightly streak into the next quadrant without overlapping the previous streak.

5) Step 3 and 4 was repeated and streak into the third quadrant.

6) Each plate was sealed with parafilm. 

7) The plates inverted and incubate at 37oC for 24 hours.

(B) Effect of handwashing on bacteria on thumb

1. 4 nutrient agar was obtained and label them:

            a) control
            b) water
            c) hand sanitizer
            d) soap

1. Each agar plate was divided into 4 sections by drawing line using a marker pen on the back of the petri dish.
2. Gently press thumb on the control agar plate by using aseptic technique.
3. Wash hands (including thumb) with water and repeat step 3 on the appropriate agar.
4. Step 4 was repeated using hand sanitizer and soap. 
5. Each plate was sealed with parafilm.
6. The plates was inverted and incubate at 37^oC for 24 hours.

Part 2: gram staining

1. Using a sterile inoculating loop, 1 drop of sterile water was add to the slide.
2. A smear of  Escherichia coli was prepared.
3. Air dry and Heat fix.
4. The smear was covered with Crystal Violet (primary stain) for 1 min.
5. Wash off the slide with water.
6. Gram’s Iodine (mordant) was added for 1 min.
7. Wash with water.Decolorize with 95% ethanol. This is the "tricky" step. Stop decolorizing with alcohol as soon as the purple color has stopped leaching off the slide (time will vary depending on thickness of smear). Immediately wash with water.
8. The smear was covered with Safranin for 30 seconds.
9. Both the top & the bottom of the slide washed with water.
10. Blot the slide.
11. Using the light microscope, 100x with immersion oil view the slide. 

Result :

Part 1 : Aseptic Technique

(A) Streak plate technique

i. E.coli

ii. S.aureus

B) Effect of handwashing on bacteria on thumb

Part 2 : Gram staining

Result from microscope observation : Gram negative bacteria

Discussion :

From this experiment, we can get the result of the effect of handwashing on bacteria on thumb using agar plate. We use 4 different types of handwashing technique. First we use bare hands without wash the hands. Next, we wash hands with the water. Later, we wash hands with soap. Lastly we use some hand sanitizer. The result show that when we wash our hands with soap, it can kill the germs on our hands better than other handwashing techniques.

In Gram staining, we get the gram-negative bacteria from microscope observation. The Gram negative bacteria is in pink colour while the Gram positive bacteria is in purple colour.

Conclusion :

From the experiment that we have done, finally, we can conclude that gram staining is the method of distinguishing between gram positive and gram negative bacteria. In this experiment,we were provided some material to help us for reaching the aim of this experiment such as Crystal violet, Gram’s iodine, 95 % ethyl alcohol, safranin, and microscope slide.However, before doing the experiment, we absolutely need to pay attention on the precautions. There are several procedures that we have to do in order to avoid the error in this experiment, such as prepare smear from cultures of microorganism, heat fix the smears, place the slides on a staining rack,and so on.

References :

1) Akbar Haqi, (2019). Lab Report of Microbiology. Retrieved from https://www.academia.edu/9655093/LAB_REPORT_OF_MICROBIOLOGY_3

2) NHS. (No Date). How to wash your hands. Retrieved from https://www.nhs.uk/live-well/healthy-body/best-way-to-wash-your-hands/

3) Pelczar Michael J, Pelczar Rita M. (2019). Microbiology. Retrieved from  https://www.britannica.com/science/microbiology