LAB 4 : SOURCES OF CONTAMINATION AND INFECTIONS

INTRODUCTION

Contamination is the state of being impure or unfit for use due to the introduction of unwholesome or undesirable elements, contaminated by insects, rodents, chemicals, microbes, or other foreign particles. In food chemistry and medicinal chemistry, contamination is used to describe harmful intrusions, such as the presence of toxins or pathogens in food orpharmaceutical drugs. In forensic science, a contaminant can be fingerprints, hair, skin or DNA from first responders or from sources not related to the ongoing investigation, such as family members or friends of the victim who are not suspects. In the biological sciences, accidental introduction of contaminant can seriously distort the results of experiments where small samples are used. In cases where the contaminant is a living microorganism, it can often multiply and take over the experiment, especially cultures, and render them useless.

Do you ever wonder why you got sick? Chances are that you most likely got sick from an airborne pathogen. In other words, the germ or irritant floated from one person to another. Airborne microorganisms are usually carried on dust particles , some of them may also be carried by direct air currents. Airborne particles are a major cause of respiratory ailments of humans, causing allergies, asthma, and pathogenic infections of the respiratory tract. During a sneeze, millions of tiny droplets of water and mucus are expelled at about 200 miles per hour (100 metres per second). The droplets initially are about 10-100 micrometres diameter, but they dry rapidly to droplet nuclei of 1-4 micrometres, containing virus particles or bacteria.

Every human is colonized by billions of microorganisms. Microorganisms may live as individuals or cluster together in communities. They live in the water we drink, the food we eat, and the air we breathe. The number of normal bacterial cells that live on the body is in the region of 100 million. This number is 10 times greater than the 10 million cells that make up the human body.

Bacteria  could be divided into two categories, namely resident or transient. The resident flora consists of microorganisms residing under the superficial cells of the stratum corneum and can also be found on the surface of the skin. Resident flora has two main protective function microbial antagonism and the competition for nutrients in the ecosystem. In general, resident flora is less likely to be associated with infections, but may cause infections in sterile body cavities, the eyes, or on non-intact skin.

Transient flora (transient microbiota), which colonizes the superficial layers of the skin, is more amenable to removal by routine hand hygiene. Transient microorganisms do not usually multiply on the skin, but they survive and sporadically multiply on skin surface.The transmissibility of transient flora depends on the species present, the number of microorganisms on the surface, and the skin moisture.

OBJECTIVE

• To determine the microorganisms in the air and from healthy humans.
• To practice the correct procedure and steps for the pour plating technique.
• To compare the differences between self-made and commercial Nutrient Broth Agar.

MATERIAL AND REAGENTS

• Molten nutrient agar
• Sterile water
• Sterile petri dishes
• Sterile clinical swab
• Pipette and tips

PROCEDURE

( Refer to lab manual )

RESULTS AND OBSERVATIONS

Figure 1 : Sample from air , top : commercial ; bottom : self-made

Figure 2 : Sample from hand , top : commercial ; bottom : self-made

Figure 3 : Sample from ear , top : commercial ; bottom : self-made

Figure 4 : Sample from normal breathing , top : commercial ; bottom : self-made

Figure 5 : Sample from violent coughing , top : commercial ; bottom : self-made

Collection of colonies	Self-made nutrient agar	Commercial nutrient agar
Air	Form : Circular Elevation : Convex Size : Small Surface : Smooth Texture : Moist Colour : Pale yellow Margin : Entire	Shape : Circular Elevation : Convex Size : Small Surface : Smooth Texture : Moist Colour : White Margin : Entire
Hands	Form : Circular , Irregular Elevation : Raised Size : Small Surface : Smooth and shiny Texture : Slightly moist Colour : White Margin : Entire and endulate	Form : Circular , Irregular Elevation : Flat Size : Small , some are large Surface : Smooth and shiny Texture : Moist Colour : Yellow Margin : Entire , Endulate
Ear	Form : Circular , Irregular Elevation : Convex , Raised Size : Small and some are large Surface : Smooth and shiny Texture : Moist Colour : Yellow and white Margin : Entire	Form : Circular , Irregular Elevation : Raised , Convex Size : Small and some are large Surface : Smooth and shiny Texture : Moist Colour : Yellow and white Margin : Entire
Normal breathing	Form : Circular Elevation : Raised Size : Small Surface : Smooth and shiny Texture : Dry Colour : Yellow Margin : Entire	Form : Circular Elevation : Raised Size : Small Surface : Smooth and shiny Texture : Dry Colour : Pink Margin : Entire
Violent coughing	Form : Circular Elevation : Raised Size : Small Surface : Smooth and shiny Texture : Dry Colour : Pink Margin : Entire	Form : Circular Elevation : Umbonate Size : Small Surface : Smooth and shiny Texture : Dry Colour : Yellow Margin : Entire , Endulate

DISCUSSION

Figure 6 : Colony morphology chart 

Bacterial populations grow extremely fast under the desired nutrients and environmental conditions. Different types of bacteria will produce colonies that are distinctive in appearance in terms of colours, shapes and sizes. In this experiment, all the aspects including form, elevation, size, surface, texture, colour and margin are studied. The differences between self-made nutrient agar and commercial nutrient agar are compared based on the morphology observed.

What we observed from the media contaminated by air is that in overall, the colonies are the fewest compared to the other media prepared. For the self-made agar media, there are a few small and circular colonies with different sizes. The colonies formed are pale yellow in colour. As for the commercial nutrient agar, only one circular colony is observed and it is white in colour.

As for the media contaminated by hand, the colonies formed are much more than what we observed by air. From here, we may assume that our hands actually contains more microorganisms or microbes than the air because we are always in contact with lots of stuff in our daily life. For the self-made agar media, it is less moist than the commercial agar media. Both of the media contains circular and some irregular bacterial colonies.

For ear contamination, the sample was taken by an ear pick. The sample is collected from the outer part of our ear and is spread on the culture by using the streaking plate technique. For the commercial nutrient agar media, it contains more colonies than the self-made nutrient agar. For the commercial nutrient agar media , it contains colonies which are mostly circular and yellow coloured colonies throughout the whole plate, being concentrated in the centre of the plate. On the other hand, for the self-made nutrient agar media, it also contains small, circular and yellow coloured colonies but are more concentrated on the sides of the plate.

For violent coughing, the colonies that could be observed is also a little, especially for the self-made nutrient agar media. Only spores like size of circular microbes can be observed under a dry texture. As for the commercial nutrient agar, a few larger sizes of circular colonies can be seen and they are yellow in colour.

The colonies observed for normal breathing is also less, there are only a few spots of colonies formed. For the self-made nutrient agar media, a yellow circular colony can be seen under a dry texture. As for the commercial nutrient agar media , a pink circular colony is seen.

CONCLUSION

In conclusion, it is important to learn the correct steps for the pour plating technique and streaking plate technique to isolate the colonies. As we can see, there are no obvious or significant differences between the colonies that grow in a self-made nutrient agar media and a commercial nutrient agar media. We also learn that it is actually important to learn about colony morphology because this is one of the ways to actually help us differentiate between different colonies and helps us in identification.

REFERENCE

1 . Obtained from https://en.wikipedia.org/wiki/Contamination on 1 November 2015.

2 . Obtained from http://www.ncbi.nlm.nih.gov/books/NBK144001/ on 1 November 2015.

LAB 3 :PREPARATION AND STERILIZATION OF CULTURE MEDIA

INTRODUCTION

Microorganisms need nutrients, a source of energy and certain environmental conditions in order to grow and reproduce. In the environment, microbes have potential to adapt to the habitats most suitable for their needs, but then it is hard for microbes to do so  in the laboratory. This is when a growth media or also known as culture media plays an important part.  This is basically an aqueous solution to which all the necessary nutrients have been added, depending on the type and combination of nutrients, different categories of media can be made. A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells. Different types of cells prefers different type of media, depending on their needs. There are actually two major types of culture media, one of it are those used for cell culture, which use specific cell types derived from plants or animals. The second one is microbiological culture, which are used for growing microorganisms, such as bacteria or yeast. The most common growth media for microorganisms are nutrient broths and agar plates.

The composition of self-made agar broth is listed below :

1.5 g/L “Lab-lemco” powder (a beef extract)

1.5 g/L Yeast extract

5.0 g/L Peptone (a nitrogen source)

5.0 g/L Sodium chloride

15.0 g/L Agar powder

For your information, the self-made agar broth actually contains the same composition with the manually-made nutrient medium, except that it contains 15 g/L agar. We must also ensure that the final pH value of both medias is 7.4 .

Speaking of autoclaving, autoclaves are more or less like pressure cookers very similar to the ones that you see in the stores. As we know, food cook a lot faster in a pressure cooker than they do in a regular pot or in the oven. This is due to the intense heat and pressure that is applied to the food. The same mechanism works against living microorganisms in an autoclave. Once an autoclave is started, steam is pushed into the chamber that contains the items that are being sterilized. As the steam goes in, the pressure and temperature within the chamber is increased. Most autoclaves are set to increase steam pressure until a temperature of at least 121 degrees Celsius is reached. This temperature and pressure will remain at this level for at least 15 minutes. This is a high enough temperature for a long enough period of time to kill any and all microorganisms and their spores. The steam and pressure are released and brought down to normal room temperature and pressure after the 15 or more minutes of running.

     Image 1 : Example of culture media in agar plates

OBJECTIVE

To prepare sterile nutrient agar for culturing microorganisms .

MATERIALS AND REAGENTS

Commercial Nutrient Agar

Brain Heart Infusion Broth ( BHI )

Trypticase Soy Broth ( TSAYE )

Peptone powder

Beef extract powder

Sodium chloride

Yeast extract

Electronic Weighing Balance

Distilled water

Scott bottles

Measuring cylinder

Glass rod

Beakers

PROCEDURE

A . Commercial nutrient agar

1 . 11.2 g of the commercial nutrient agar is weighted using an electronic

    weighing balance and placed into a beaker.

2 . 400 ml of distilled water is measured using a measuring cylinder and poured into

    the beaker containing the nutrient agar. The solution is then stirred by using a

     glass rod until it mixes well.

3 . After the solution is mixed well, the solution is poured into the Scott bottle that

    had been sterilized.

4 . The bottle is the loosely recapped and is set aside and ready to undergo

    sterilization in an autoclave machine.

5 . The media is sterilized at 121 degree Celsius for 15 minutes.

6 . The media is removed after 15 minutes of autoclaving. The media is allowed to

     cool down and the cap of the bottle is tighten.

B . Self-made nutrient agar

1 . 0.60 g of beef extract, 0.6 g of yeast extract, 2.0 g of peptone, 2.0 g of sodium

    chloride and 6.0 g of agar powder are weighed using an electronic weighing

    balance and placed into a beaker.

2 . 400 ml of distilled water is measured using a measuring cylinder and poured into

    the beaker containing the nutrient agar. The solution is then stirred by using a

    glass rod until it mixes well.

3 . After the solution is mixed well, the solution is poured into the Scott bottle that

    had been sterilized.

4 . The bottle is the loosely recapped and is set aside and ready to undergo

    sterilization in an autoclave machine.

5 . The media is sterilized at 121 degree Celsius for 15 minutes.

6 . The media is removed after 15 minutes of autoclaving. The media is allowed to

     cool down and the cap of the bottle is tighten.

C . Brain Heart Infusion agar (BHI)

1 . 5.20 g of BHI agar in powder form is weighed using an electronic weighing

    balance and placed into a beaker.

2 . 100 ml of distilled water is measured using a measuring cylinder and poured into

    the beaker containing the nutrient agar. The solution is then stirred by using a

    glass rod until it mixes well.

3 . After the solution is mixed well, the solution is poured into the Scott bottle that

    had been sterilized.

4 . The bottle is the loosely recapped and is set aside and ready to undergo

    sterilization in an autoclave machine.

5 . The media is sterilized at 121 degree Celsius for 15 minutes.

6 . The media is removed after 15 minutes of autoclaving. The media is allowed to

     cool down and the cap of the bottle is tighten.

D . Trypticase Soy Agar ( TSAYE )

1 . 4.00 g of TSAYE agar in powder form is weighed using an electronic weighing

    balance and placed into a beaker.

2 . 100 ml of distilled water is measured using a measuring cylinder and poured into

    the beaker containing the nutrient agar. The solution is then stirred by using a

    glass rod until it mixes well.

3 . After the solution is mixed well, the solution is poured into the Scott bottle that

    had been sterilized.

4 . The bottle is the loosely recapped and is set aside and ready to undergo

    sterilization in an autoclave machine.

5 . The media is sterilized at 121 degree Celsius for 15 minutes.

6 . The media is removed after 15 minutes of autoclaving. The media is allowed to

     cool down and the cap of the bottle is tighten.

RESULTS

4 different culture media was prepared which are 400 ml of commercial nutrient agar, 400 ml of self-made nutrient agar, 100 ml of Brain Heart Infusion (BHI) agar and 100 ml of Trypticase Soy Agar ( TSAYE ). The composition of the materials needed are stated in the procedure. They are weighed approximately and dissolved with distilled water. Stirring of the solution takes place until the solution is dissolved and mixed. After mixing, it is only poured into sterilized Scott bottles and ready to place into an autoclave machine for 15 minutes at the temperature of 121 degree Celsius.

Image 2 : Weighting the appropriate amount using an electronic weighting balance

Figure 3 : Done weighing the 6.00 g agar powder for self-made nutrient agar

Figure 4 : The preparation for self-made nutrient agar

Figure 5 : Appropriate amount of distilled water obtained using a measuring cylinder

Figure 6 : Transfer of the solution after mixing with distilled water into Scott bottles 

Figure 7 : Four different cultured media prepared

Figure 8 : Cultured media ready to undergo autoclaving

Figure 9 : Cultured media in an autoclave

DISCUSSION

1 . There are actually a few precautions that we need to take note throughout the

     experiment, which is :

• The pan of the electronic weighing balance is cleaned with a small brush first to prevent any small leftover particles that might affect the weight reading.
• The “tare” button is pressed every time after the empty beaker is put on the balance to obtain accurate measurements and to prevent zero errors.
• When using a measuring cylinder to obtain distilled water, make sure that the position of the eye is at the same level as the bottom of the meniscus ( surface of water that is curved downwards ) to prevent parallax errors.
• All of the apparatus used are cleaned and rinsed using distilled water before using.
• The media is stirred well using a glass rod to ensure balance mixing and to maintain the concentration of the media.
• Make sure that the caps of the Scott bottles are only slightly tightened to prevent the Scott bottles from breaking during autoclaving.

2 . Before the Scott bottles with different medium are placed into the autoclaving machine for sterilization, there are some steps that need to be followed as below :

• The drain screen at the bottom of the chamber is checked before using the autoclave.
• Any debris noticed is cleaned up for efficient heat transfer as steam must flush out of the autoclave chamber. If the drain screen is blocked with debris, a layer of air may form at the bottom of the autoclave and prevent proper operation.
• The water level is ensured to be higher than the bottles in the autoclave.  
• The cover of the autoclave chamber and exhaust valve is tightened.
• The temperature is checked so it is always maintained at 121°C and the pressure is ensured to reach 103 kPa above the atmospheric pressure, with steam is continuously forced into the chamber.
• The time for destruction of the most resistant bacterial spore is now reduced to about 15 minutes. For denser objects, up to 30 minutes of exposure may be required. The conditions must be carefully controlled or serious problems may occur.
• Then, the exhaust valve is opened to ensure the pressure drops to nearly 0 kPa before removing the basket with Scott bottles from the autoclave chamber.

CONCLUSION

As a conclusion, we are able to learn the correct steps and methods to prepare different media for culturing microorganisms. Precautions must also remembered when carrying out the steps and procedures.  Preparation and sterilization of culture media is important to prevent contamination of the unwanted microorganisms inside the media. We also obtained the information that autoclaving is actually a fast and efficient sterilization process.   

Reference

1 . Obtained from https://en.wikipedia.org/wiki/Growth_medium on 24 October 2015

2 . Obtained from

http://www.sigmaaldrich.com/analytical-chromatography/microbiology/learning-center/theory/media-preparation.html on 24 October 2015

3. Obtained from

  http://study.com/academy/lesson/what-is-an-autoclave-definition-uses.html on 24

  October 2015

4. Obtained from https://en.wikipedia.org/wiki/Autoclave on 24 October 2015