BIO300/NOV 2013

UNIVERSITI TEKNOLOGI MARA NEGERI SEMBILAN

FACULTY OF APPLIED SCIENCES

BIOLOGICAL TECHNIQUES AND SKILLS

BIO300

PROJECT PROPOSAL

AS1205A2 TITLE: THE EFFECTIVENESS OF SHOWER

CREAMS TOWARDS BACTERIA IN BODY SKIN.

BIO300/NOV 2013

PART A: RESEARCHER BACKGROUND

i. PROJECT LEADER/ MATRIC NO. /CONTACT NO.:

MOHD SHAHIRUL BIN MD ISA

2011256166 017-3404196

ii. PROJECT MEMBER’S:

NO. NAME MATRIC

NUMBER

CONTACT

NUMBER

1. RASEM SHAH BIN KARAB HUSSAIN

2011753195 012-6524722

iii. PROJECT SUPERVISOR:

SYAKIRA BINTI MOHAMMED HUSSEIN

iv. LECTURER (Lecture/Laboratory)

IZZATI ADILAH BINTI AZMIR

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PART B: RESEARCH PROJECT PROPOSAL

i) OBJECTIVES:

(Minimum of 2 objectives, maximum of 4 objectives)

1. To indentify which body parts contain more amounts of bacteria by colony

counting method.

2. To investigate which shower creams are more effective towards various

types of bacteria.

ii) SIGNIFICANCE OF PROJECT:

(why this project is important? Benefits to the students, society etc.)

This project is most important because to prove the effectiveness of

shower cream towards the various types of bacteria on human body. Most of

human being uses the shower cream as antibacteria agent in daily life but

still unconscious about the effectiveness of shower creams. Furthermore,

this project is to help the society to choose the best shower creams that can

help against the various types of bacteria. Besides that, this project can give

impact to athletes or university students who are active in sports to choose

the best shower creams for those athletes to take after doing their activity.

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iii) DEFINATION OF TERMS/CONCEPTS:

(any related scientific terms or concepts that need elaboration)

1. Staphylococcal bacteria: Gram-positive bacteria, genus of micrococci

bacteria with many members that can cause disease.

2. Escherichia coli (E. coli): Gram-negative bacteria that contain strains that

are harmless but can cause serious food poisoning in humans.

3. Klebsiella: Gram-negative bacteria that can cause different types of

healthcare-associated infections, including pneumonia, bloodstream

infections, wound or surgical site infections, and meningitis.

4. Serratia marcescens: Species of Gram-negative, rod-shaped bacterium in

the family Enterobacteriaceae. S. marcescens is involved in nosocomial

infections particularly catheter-associated bacteremia.

5. Bacillus sp.: Bacillus species are rod-shaped, endospore-forming aerobic

or facultatively anaerobic, Gram-positive bacteria; in some species

cultures may turn Gram-negative with age.

6. Nutrient agar: Nutrient agar is a microbiological growth medium commonly

used for the routine cultivation of non-fastidious bacteria. It is useful

because it remains solid even at relatively high temperature.

7. Mueller- Hinton agar: Medium containing beef infusion, peptone and

starch used primarily for the disk- agar diffusion method for antimicrobial

suspectibility testing.

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iv) LITERATURE REVIEW:

(The information of previous studies on the related project. Please select

recent and reputable publication. Recent publication may subject to the

nature of field and the accessibility of the information. The use of

Endnote or other bibliography software as referencing tools is

encouraged. Add more pages if necessary.)

There are many bacteria live in human body skin. One of them is

Staphylococci Aureus. Staphylococci are one of the clinical bacteria that exist

around us. To be more specific, staphylococci are Gram-positive bacteria that

are characterized by individual cocci, which divide in more than one plane to form

grape-like clusters (Harris & Foster, 2002). These bacteria got its name from the

Greek, staphylē which means grapes because of its shape that is more likely to

the fruit. The staphylococci are non-motile, non-spore forming facultative

anaerobes that grow by aerobic respiration or by fermentation. This type of

bacteria is tolerant to high concentrations of salt (Lowy, 2013) and show

resistance to heat.

Next, Escherichia coli (E. coli) are a Gram-negative, facultative anaerobic,

rod-shaped bacterium that is commonly found in the lower intestine of warm-

blooded organisms (endotherms). Most E. coli strains are harmless, but some

serotypes can cause serious food poisoning in humans, and are occasionally

responsible for product recalls due to food contamination. The harmless strains

are part of the normal flora of the gut, and can benefit their hosts by producing

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vitamin K2, and by preventing the establishment of pathogenic bacteria within the

intestine.

Thirdly, Klebsiella is a genus of non-motile, Gram-negative, oxidase-

negative, rod-shaped bacteria with a prominent polysaccharide-based capsule. It

is named after the German microbiologist Edwin Klebs (1834–1913). Frequent

human pathogens, Klebsiella organisms can lead to a wide range of disease

states, notably pneumonia, urinary tract infections, septicemia, and soft tissue

infections. Klebsiella species have also been implicated in the pathogenesis of

ankylosing spondylitis and other spondyloarthropathies.

Fourth, Serratia marcescens is a species of rod-shaped Gram-negative

bacteria, in the family Enterobacteriaceae. A human pathogen, S. marcescens is

commonly found in the respiratory and urinary tracts of hospitalized adults and in

the gastrointestinal system of children (Brock, 1984). Due to its abundant

presence in the environment, and its preference for damp conditions, S.

marcescens is commonly found growing in bathrooms especially on tile grout,

shower corners, toilet water line, and basin, where it manifests as a pink

discoloration and slimy film feeding off phosphorus-containing materials or fatty

substances such as soap and shampoo residue. S. marcescens may also be

found in environments such as dirt, supposedly "sterile" places, and the

subgingival biofilm of teeth. S. marcescens may cause extrinsic staining of the

teeth.

Bacillus sp. is a genus of Gram-positive, rod-shaped (bacillus), bacteria and

a member of the phylum Firmicutes. Bacillus species can be obligate aerobes

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(oxygen reliant), or facultative anaerobes (having the ability to be aerobic or

anaerobic). Some types of Bacillus bacteria are harmful to humans, plants, or

other organisms. For example, B. cereus sometimes causes spoilage in canned

foods and food poisoning of short duration. B. subtilis is a common contaminant

of laboratory cultures and is often found on human skin (Wood W., 1993). Most

strains of Bacillus are not pathogenic for humans but may, as soil organisms,

infect humans incidentally. A notable exception is B. anthracis, which causes

anthrax in humans and domestic animals. B. thuringiensis produces a toxin (Bt

toxin) that causes disease in insects.

An important fact to be kept in mind is these bacteria can cause disease in

humans and animals are not inherently pathogenic organisms – unlike the

influenza virus, for example, which must infect to propagate. These bacteria is an

organism that colonizes the skin, particularly the anterior nares, skin folds,

hairline, perineum and umbilicus. It commonly survives in these areas without

causing infection – a state known as colonization. A patient becomes clinically

infected if the organism invades the skin or deeper tissues and multiplies to

cause a localized or systemic response, for example in septicaemia.

These bacteria can be transmitted by endogenous spread and exogenous

spread. Endogenous spread occurs when a person with bacteria spreads the

bacteria from one part of their body to another while exogenous spread occurs

when organisms are transferred from person to person by direct contact with the

skin or via contaminated environments or equipment (P. R. Murray, 1995). Skin

scales may contaminate all surfaces if they become airborne, for example during

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activities such as bed making, or if the affected person is heavily colonized, or

has a condition such as eczema which causes skin shedding which will result in

widespread distribution of many skin organisms.

To prevent from this kind of infection, there are some precautions that are

essential to be taken in order to against the infection that is clearly caused by

these bacteria. According to P.R. Murray (1995), we should cover all cuts,

abrasions and lesions that are especially those on hands and forearms with a

waterproof dressing. Besides that, we are advised to maintain hand hygiene

before and after each patient contact and also after handling blood and body

fluids and items contaminated with blood and body fluids. Maintaining the

cleanliness of general environment – horizontal surfaces, floors, toilets, sinks,

baths (and walls if soiled) is also important.

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v) REFERENCES:

(at least 10 recent and reputable references)

Causes. (n.d.). Retrieved July 5, 2013, from http://www.mayoclinic.com/:

http://www.mayoclinic.com/health/staphinfections/DS00973/DSECTION=causes

Guzmán-Blanco, M., Mejia, C., Isturiz, R., Alvarez, C., Bavestrello, L., Gotuzzo,

E., et al. (2009). Epidemiology of meticillin-resistant Staphylococcus aureus

(MRSA) in Latin. International Journal of Antimicrobial Agents , 304-308.

Harris, L., Foster, S. J., & Richards, R. G. (2002). An introduction to

staphylococcus aureus and techniques for identifying and quantifying s.aureus

adhesins in relation to adhesion to biomaterials. European Cells and Materials,

56-59.

Lowy, F. D. Staphylococcal infections. Methicillin-resistant Staphylococcus

aureus (MRSA) Infection. (2013). Virginia Department of Health . retrieved

July5,2013,fromhttp://www.nhs.uk/conditions/mrsa/documents/rcn%20mrsa%20

guidelines.pdf

Staph bacteria: Sorting through the hype. (n.d.). Hirsch Pediatrics . retrieved July

5, 2013,from http://www.hirschpediatrics.com/documents/ Staphbacteria_

Sortingthrough thehype.pdf

P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (eds.).

(1995) Manual of clinical microbiology, 6th ed. American Society for

Microbiology, Washington, D.C.

Brock, T.D., D.W. Smith and M.T. Madigan, (1984). Biology of Microorganisms.

4th Edn., Prentice-Hall International Inc., New Jersey, USA..

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Boboye, B., T. Babatunde and A. Onoriode, (2007). Antibacterial activities of

some plants used as condiments and spices in Nigeria. World Curr. Environ., 5-

8.

Personne, P., M. Bes, G. Lina, F. Vandenesch, Y. Brun, and J. Etienne, (1997).

Comparative performances of six agglutination kits assessed by using typical and

a typical strains of Staphylococcus aureus. J. Clin. Microbiology, 2-3.

Wood, W., G. Harvey, E. S. Olson, and T. M. Reid, (1993). Aztreonam selective

agar for Gram positive bacteria. J. Clin. Pathol, 2-4.

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vi) RESEARCH METHODS:

(add more pages if necessary)

Sterilization of Apparatus

1. Place cotton wool plugs in each test tubes and cover plugs loosely with

aluminium foil.

2. Place a small piece of cotton wool on each of eight 1mL graduated pipettes

and the 10µL graduated pipette and wrap each separately in aluminium foil.

3. Place the test tube and pipettes in a hit oven at 120 ºC for 15 minutes (bottle

of media and water should not be sterilized in an autoclave).

4. Dip the glass spreader in 70% alcohol,allow alcohol excess to drip off and

hold the spreader vertically in a Bunsen burner flame.

5. Allow all apparatus to dry before use.

Preparation of Nutrient Agar

1. To weigh out Beef Extract, first tare a tongue depressor, then dip it into the

Beef Extract and weigh. Adjust the amount of Beef Extract until the correct

amount is obtained. Be sure to be careful not to get Beef Extract on to the

balance! Weigh enough Beef Extract to get a 0.3% solution. Place the

tongue depressor into the flask, beef extract side down.

2. Tare a weigh boat and weigh out enough Peptone and add that to the flask.

3. Add 200 ml of distilled water and swirl to dissolve the peptone and beef

extract. Check the pH, it should be 7.0.

4. Tare a weigh boat and weigh out enough Agar and add that to the flask.

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5. With a bunsen burner, tripod, asbestos wire-gauze, heats the medium to

boiling to dissolve the agar. CAREFUL: 1) keep the rotating the flasks to

prevent the agar from cooking onto the bottom of the flask and 2) watch out:

boiling agar can froth and boil out all over the lab bench. As soon as it begins

to boil take it off the heat and put it on to the bench. Allow it to cool a few

minutes.

6. While the agar is still warm, but not hot, pipette 3 ml each into 4 13x100 mm

screw cap culture tubes.

7. Label the flask and your tubes with your name.

8. After preparation of medium, take to the autoclave.

9. Place the media in the autoclave with those of the rest of the class.

10. After discussion of the parts of the autoclave, autoclave the medium 120oC

for 20 minutes.

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Collecting. Identification and Preparation Bacterium

1. Label bottles and Petri dishes with A1, A2, A3, B1, B2, and B3.

2. Collect bacterial sample from nacres, knuckles, forearms, and shoes by

using sterile cotton buds before and after playing football.

3. Put the cotton buds in the bottles that contain nutrient broth for more than 14

hours.

4. Using the serial dilution method to prepare a series of dilutions to a known

factor.

5. Swab the dilutions on each petri dish that contain Nutrient agar. Put the petri

dishes in the incubator for 1 day. If the colour of the agar changes, proceed

to the preparation of Staphylococcus bacteria.

6. From the Petri dishes that contain Staphylococcus bacteria, prepare a new

nutrient broth for another types bacteria growth (E. coli, Klebsiella, Seratia

Mercescens, Bacillus sp.). Put in the incubator for 1 day.

Serial Dilution of Bacteria

1. Label the six sterile plugged test tubes as S1 to S6.

2. Transfer 9 mL of the nutrient broth to each of the six test tubes using the

following technique:

a) Remove the bottles cap containing the nutrient broth

b) Draw up 9 mL of nutrient broth using the sterile 10 mL graduated pipettes

c) Replace the bottles cap.

d) Remove the plug from the first test tube (S1)

e) Transfer the 9 mL of nutrient broth to the test tube

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f) Replace the test tube plug.

g) Repeat the process for the other five remaining test tubes

3. Using a fresh tip, transfer 1 mL from tube S1 to the next tube S2 and repeat

step 3 until the last tube, S6.

Determine the bacterial population sensitivity to a range of shower creams.

1. Place small disks of filter paper or agar impregnated with various types of

shower creams onto the bacterial lawn.

2. Put the bacteria in incubator for 1 day.

3. Then, the plate is examined to see whether the bacterial growth is inhibited

(or not) by the shower creams on each disk.

SENSITIVE: In this case, a clear, circular "halo" (technically known as a

"plaque” or zone of inhibition) will appear around the antibiotic disk,

indicating an absence of bacteria. The antibiotic has inhibited their growth

and/or killed them, meaning that this particular antibiotic should be

effective against the infection your rabbit has.

INTERMEDIATE: A somewhat cloudy plaque indicates that not all the

bacteria in the area around the disk have been killed. This means that

there are some members of the bacterial population that are sensitive to

this particular antibiotic, but others that are genetically immune to its

effects. If an antibiotic to which the bacteria show "intermediate"

sensitivity is used, it is likely that the sensitive members of the bacterial

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population will be killed, and the resistant ones will survive, resulting in

the selection of a population resistant to that particular antibiotic.

RESISTANT: In this case, the filter paper will have no discernible plaque

around it, meaning that the bacteria are growing normally, even in the

presence of the antibiotic. An antibiotic producing no plaque will most

likely be ineffective against the bacteria causing your bunny's infection.

4. Measure the diameter zone of inhibition in mm by using a ruler.

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vii) LIST OF EQUIPMENTS/FACILITIES:

1. Cotton buds.

2. Aluminum foil.

3. 1 mL micropipettes.

4. 200 μL micropipettes.

5. 10 mL graduated pipettes.

6. Measuring Cylinder.

7. Glass spreader.

8. Petri dish.

9. Test tubes.

10.Beaker.

11.Auto clave.

12.Ruler.

13. Incubator.

14.Dropper.

15. Specimen bottles.

16. Universal bottle.

17. Stopper.

18. Spatula.

19. Bunsen burner.

20. Tripod.

21. Wire gauze.

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viii) LIST OF CHEMICALS/MEDIA:

1. Various brands of shower cream.

2. Staphylococus Aureus bacteria.

3. E. coli bacteria.

4. Klebsiella bacteria.

5. Serratia marcescens bacteria.

6. Bacillus sp. bacteria.

7. Beef extract.

8. Peptone.

9. Nutrient broth.

10.Mueller – Hinton Agar.

11.Tissue paper.

12.Glove.

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ix) DECLARATION AND SIGNATURE

“We are hereby declaring that the project is based on our original

work except for quotations and citations which have been duly

acknowledge. We are conducting our investigations with honesty

and integrity”

SIGNATURE OF PROJECT LEADER: ________________________

(MOHD SHAHIRUL BIN MD ISA)

SIGNATURE OF PROJECT MEMBER:

1. RASEM SHAH BIN KARAB HUSSAIN : _________________________

Signature of Supervisor: _________________________________

Received date: ____________________________

Returned date: ____________________________