LAB 6 : MICROBIOLOGY

Part 1: Aseptic Technique

1.0      OBJECTIVE          
1)  To produce isolated colonies of an organism on an agar plate and useful when we need to seperate organisms in a mixed cultures
2)  To identify bacteria are only valid when performed on pure cultures.

1.1     INTRODUCTION

Bacteria are everywhere, and some are good for us while others are harmful. Bacteria, viruses, and other microorganisms that cause disease are called pathogens. To protect patients from harmful bacteria and other pathogens during medical procedures, healthcare providers use aseptic technique.

An acid/base titration can be monitored with an indicator or with a pH meter. In either case, the goal is to determine the equivalence point of the titration. This is the point at which enough titrant has been added to the analyte to just exactly neutralize the analyte.

In this experiment, knowledge of the equivalence point will be used to obtain information about the acid dissociation constant, Ka, of the acid being titrated. When an indicator is used in a titration, the color change occurs at what is called the endpoint. If the indicator has been properly selected, this point will be the same as the equivalence point. When a pH meter is used, the pH of the solution is recorded as the titrant is added. The pH versus the volume of titrant added can be plotted on what is called a titration curve. In this case the equivalence point occurs at the point where very small additions of titrant cause a very rapid rise in the pH.

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.

1.3 MATERIALS/ APPARATUS
1.Bacteria
2. Loop
3. Bunsen burner
4.Alcohol
5. Agar plate 
6. Ethane

1.4       METHODOLOGY

(A) Streak plate technique

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

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

3.  The agar plate was cover 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 seales with parafilm.
7.  The plates was inverted and incubated at 37^oC for 24 hours.

(B) Effect of handwashing on bacteria on thumb

1.  4 nutrient agar and label them:

a)  control

b)  water

c)  hand sanitizer

d)  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.  Aseptic technique was applied, press thumb on the control agar plate gently.

4.  Hands was washed (including thumb) with water and step 3 was repeat on the appropriate agar.

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

6.  Each plate was sealed with parafilm.

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

1.5       RESULTS
Part A:   Streak plate technique

Part B : Effect of handwashing on bacteria on thumb

1.6     DISCUSSION

                 The streak plate procedure, a mixture of cells is spread over the surface of an agar medium in the petri dish such that fewer and fewer bacterial cells are deposited at widely separated point on the surface of the medium and following incubation develop into colonies.  Label around edge of the bottom of an agar with at least name, the date and the type of the organism to be plated on the medium. The last process is to place the plate into incubater. The plate can not be left in more than 24 hours because the colony in the plate will spread and fill the plate.

              The plate is divide into four parts namely control, hand senitizer, water and soap. The plate has to divided because it can distinguish where the bacteria will appear with much. The proper hand washing technique is applied in the soap section to make sure the bacteria at our hand are clean as well. From the result, the hand washing experiment indicate that washing with hand sanitizer is more effective.  In addition, washing with soap appeared to be effective also but initially lagged in removing microorganisms. One reason for this lag may be that the soap was effective in loosening the microorganism at deeper layers of the epidermis.

1.7     CONCLUSION

          The conclusion is streak plate method is one of the most common method for the isolation of pure culture of all microorganism. For the effect hand washing, we can conclude that by using hand sanitizer is most effective than the other method of hand washing. 

Part 2 : Gram Staining

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2.0       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.

2.1  OBJECTIVES:

1 )   To differentiate between the two major catagories of bacteria, gram positive and gram negative.

2)      To understand how the gram stain  reaction affect gram poeitive and gram negative.

2.2 APPARATUS AND EQUIPMENTS

• Glas side
• Loop
• Bunser burner
• Sample
• Distilled water
• Alcohol
• Iodin
• Cyrstal violet
• Safranin

2.3         METHODOLOGY:

1.  A sterile used inoculating loop, 1 drop was added of sterile water to the slide. Prepare a smear of:

(a)  Escherichia coli and (b) Staphylococcus aureus

2.  Dried air and fixed the heat.

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

4.  The slide wash off with water gently.

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

6.  Wash with water.

7.   Decolorized 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. Be sure to dispose of all ethanol waste in the appropriately labelled waste container.

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

9.  Both the top & the bottom was wash of the slide with water.

10.  The slide was bloted.

11.  Light microscope used, view the smear up to 100x with immersion oil.

2.4     RESULT

FOR E.COLI

 

E.coli 4x

E.Coli 10x

E.coli 40x

E.coli 100x

FOR STAPHYLOCOCCUS AUREUS 

Staphylococcus aureus 4x

Staphylococcus aureus 10x

Staphylococcus aureus 40x

Staphylococcus aureus 100x

2.5 DISCUSSION

By using gram staining, there are two type of bacteria that exits in the slide which is staphylococcus aureus and Escherichia coli. We used the bacterial colony in the spread plate for staining.

The slide was heated because heating enables coagulation and precipitation of protein of bacteria to occur, hence fix the bacteria on slide the bacteria killed and adhere to the surface. In addition, cystal violet is added to stain everything on slide or to stain all bacteria. The extra cystal violet dye that not binds to cell is cleared by distilled water. Addition of iodine in next step anables the cystal violet dye further fix and adhere to organisms. Iodine acts as mordant as it increase affinity of cystal violet stain to organisms. The addition of ethanol as decolourizer enables the lipid to be extracted or dissolved from the cell wall from gram negative bacteria like the Escheriichia Coli. Ethanol was not added for more than 30seconds because over decolourization can cause the stain og gram positive bacteria to decolourize and appears as gram negative.

Under microscope, staphyloccus aureus is rod-shaped and apprear in purple colour. While Escherichia coli is rod-shaped and appear in red colour.

2.6     CONCLUSION

Gram staining is important in differentiating gram positive and gram negative bacteria in which the gram positive bacteria stained purple colour while gram negative organisms stained pink. Escherichia coli is gram negative while staphylococcus aureus are gram positive bacteria.   

2.7        REFERENCES

1)      Wise, K. Preparing Spread Plates Protocols (2006) . Retrived from http://www.microbelibrary.org/index.php/component/resource/laboratory-test/3085-preparing-spread-plates-protocols

2 ) Worthington, M, Luo, R. Q, Pelo, J. Method Of Seperating Organism, 30, 738-742 (2001)

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