LAB 6 : MICROBIOLOGY

LAB 6: MICROBIOLOGY

Part 1: Aseptic Technique

INTRODUCTION

The streak plate method is a rapid qualitative isolation method. The techniques commonly used for isolation of discrete colonies initially require that the number of organisms in the inoculums be reduced. It is essentially a dilution technique that involves spreading a loopful of culture over the surface of an agar plate. The resulting diminution of the population size ensures that, following inoculation, individual cells will be sufficiently far apart on the surface of the agar medium to effect a separation of the different species present.

Figure 1: Four quadrant streak on agar plate

The hands are the parts of the human body that are in most contact with the outside world. People use their hands for a variety of activities everyday. It is extremely easy to come in contact with different microbes and to transfer them to other objects and maybe even people.

Handwashing is thought to be effective for the prevention of transmission of pathogens. There are many products in the market claim to be able to kill the germs. However it is not conclusive that handwashing with soap or other cleaning products is more effective at reducing bacteria contamination than using water only.

Figure 2: the materials used in this experiment

METHODOLOGY

(A) Streak plate technique

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

2. An isolated colony was picked from the agar plate culture of (a) E. coli and (b) S. aureus and spread each of them over the first quadrant on separated agar plate.

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

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

5. Step 3 and 4 were repeated and streaked into the third quadrant.

6. Each plate with parafilm was sealed.

7. The plates were inverted and incubated at 37°C for 24 hours.

(B) Effect of handwashing on bacteria on thumb

1. 4 nutrient agar was obtained and labelled

control

water

hand sanitizer

soap

2. Each agar plate was divided into 4 sections by drawing line using a marker pen on the back of the petri dish.

3. Using aseptic technique, gently press thumb on the control agar plate.

4. Wash hands (including thumb) with water and step 3 was repeated on the appropriate agar.

5. Step 4 was repeated using hand sanitizer and soap.

6. Each plate with parafilm was sealed.

7.The plates was inverted and incubated at 37°C for 24 hours.

RESULT

(A) Streak plate technique

Figure 3:E. coli

Figure 4: S. aureus

(B) Effect of handwashing on bacteria on thumb

Figure 5: the bacteria on thumb

Part 2: Gram staining

INTRODUCTION

The Gram stain is a differential stain commonly used in the microbiology laboratory that differentiates bacteria on the basis of their cell wall structure. Most bacteria can be divided into two groups based on the composition of their cell wall:

1. Gram-positive cell walls have a thick peptidoglycan layer beyond the plasma membrane. Characteristic polymers called teichoic and lipoteichoic acids stick out above the peptidoglycan and it is because of their negative charge that the cell wall is overall negative. These acids are also very important in the body’s ability to recognize foreign bacteria. Gram- positive cell walls stain blue/purple with the Gram stain.

2. Gram-negative cell walls are more complex. They have a thin peptidoglycan layer and an outer membrane beyond the plasma membrane. The space between the plasma membrane and the outer membrane is called the periplasmic space. The outer leaflet of the outer membrane is composed largely of a molecule called lipopolysaccharide (LPS). LPS is an endotoxin that is important in triggering the body’s immune response and contributing to the overall negative charge of the cell. Spanning the outer membrane are porin proteins that enable the passage of small molecules. Lipoproteins join the outer membrane and the thin peptidoglycan layer. Gram-negative cells will stain pink with the Gram stain.

METHODOLOGY:

1. Using a sterile inoculating loop, 1 drop of sterile water was added to the slide. A smear were prepared.

Escherichia coli

Staphylococcus aureus

2. Air dried and Heated fix.

3. The smear was covered with Crystal Violet (primary stain) for 1 min.

4. Gently washed off the slide with water.

5. Gram’s Iodine (mordant) was added for 1 min.

6. Washed with water.

7. 95% ethanol was decolorise. This is the "tricky" step. Stopped decolorizing with alcohol as soon as the purple color has stopped leaching off the slide (time will vary depending on thickness of smear). Immediately washed with water. Be sure to dispose of all ethanol waste in the appropriately labelled waste container.

8. The smear was covered with Safranin for 30 seconds.

9. both the top & the bottom were washed of the slide with water.

10. Blotted the slide.

11. Using the light microscope, the smear was viewed up to 100x with immersion oil.

RESULT

(a) Escherichia coli

Figure 6: the smear at 40x

Figure 7: the smear up to 100x with immersion oil.

(b) Staphylococcus aureus

Figure 8: the smear at 40x

Figure 9: the smear up to 100x with immersion oil.

DISCUSSION

In this experiment, we focused on gram staining and the mechanisms in gram staining. As usual, before we started the experiment, it is crucial for us to perform aseptic technique In order to minimize contamination. Firstly, we prepared smears of two microorganisms which are Bacillus anthracis and Escherichia coli.

In theory, both of this microorganisms are from two different groups, and we expected to observe two different result in this gram staining experiment. After preparing the smears of the microorganisms, the smears were air dried and heat fixed. Then we stain both smears using crystal violet dyes. Crystal violet dyes are basic dyes that have positively charged particle that helps them to bind to negatively charged molecule like teichoic acid at the cell wall of bacteria. This crystal violet dye can dissociate into cv+ and cv- ions. These ions can penetrate deeps into the cell wall of bacteria and interacts with the negatively component on the bacterial cell wall. 1 minute later, the crystal violet was washed with tap water and then the slides are dried. The next step is to add iodine onto each smear. Iodine was being added as a mordent to form crystal violet-iodine complex, CVI complex. This complex enables the dyes to not be easily being removed. Next, we washed the iodine with tap water and dried off the excess water. After that, 95% of ethyl alcohol was being added to acts as a decolorizing agent. It interacts with the lipid membrane of both positive and negative bacteria, and this would cause the gram-negative bacteria to lost their outer membrane and exposing the peptidoglycan. The CVI complexes are being washed from the outer membrane of gram-negative bacteria and cause the purple colour to decolorize. Meanwhile, for gram-positive bacteria, the addition of alcohol dehydrated the layer of peptidoglycan which in turn would trap the CVI complex. This cause the gram positive bacteria appeared to be purple colour as the CVI complex are being retained. The addition of alcohol is not be more than 15 seconds as this would break the cell wall of the bacteria, thus resulting in no stain to be observed. The slides are washed with water and dried off. In the next step, safranin was used to counterstain both smears. This is to enables the gram negative bacteria to be visualized easily as it can be stain in pink colour. The gram-positive bacteria does not being stained pink when safranin was being introduced because the peptidoglycan layer already have CVI complex. Then, the slides were washed using tap water and dried off. Finally, we observed our specimen using microscope under oil-immersion objective lenses. These particular lenses have more mirrors inside and it requires the use of oil to refract light rays towards the center of the lenses.

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.

Most of the microbes in humans live a harmonious existence with human cells, but disease and infection can be caused when this balance is disrupted or when the body or immune system is weakened. Microbes can transfer genetic information between one another and this is one of the means by which antibiotic resistance is spread among the microbial population. The process is the origin of MRSA.

REFERENCES

Sonya Dougal, PhD, (2019), Microbiology and Infectious Diseases Discussion Group, retrieved from,
https://www.nyas.org/programs/frontiers-of-science/microbiology-and-infectious-diseases-
discussion-group/

DhayaKutty, (2008), microbiology, retrieved from,

http://microbgy.blogspot.com/2008/02/conclusion.html

PROJECT RESULT

From our project, we used water cooler, distilled water, mineral water and tape water to observe some bacteria that can grow and the most in water. From our observation, we can see there are a lot of bacteria in distilled water while a little bit in water cooler. This are shown water cooler more save to drink.