Objectives: 1. To identify the human normal flora at six different places. 2.

To observe their biochemical reaction by doing biochemical tests. Introduction: The human flora is the assemblage of microorganisms that reside on the surface and in deep layers of skin, in the saliva and oral mucosa, and in the gastrointestinal tracts. They include bacteria, fungi and archaea. Some of these organisms are known to perform tasks that are useful for the human host, while the majority have no known beneficial or harmful effect. Those that are expected to be present, and that under normal circumstances do not cause disease, but instead participate in maintaining health, are deemed members of the normal flora, or microbiota. Body is host to billions of bacteria of many different kinds. These bacteria exist in many different parts of the body, and usually do not cause any problems for the host body. The following is a list of the main sites for bacteria that consitute the normal flora.

The skin, especially the moist areas, such as the groin and between the toes. The respiratory tract, particularly the nose. Urinary tract. The digestive tract, i.e. the mouth, the terminal ileum and the colon.

There are many different types of relationship that the body can have with the normal flora. These are

Mutualism. In a mutualist relationship, both the host and the microbe benefit from the relationship. The best example if this is E.Coli. This organism lives in the intestines, where it receives nourishment, and in turn produces Vitamin K, which the human body requires for the process of blood clotting.

Commensalism. A commensalist relationship is where one partner of the relationship benefits and the other partner are neither benefitted nor harmed.

Parasitism. A parasitic relationship is where one organism benefits at the expense of the host. The cost to the host can vary from slight to fatal. An external parasite (ectoparasite) is said to cause infestation, an internal parasite (endoparasite) is said to cause infection.

Pathogenic. A pathogenic relationship is where an organism causes damage to the host during infection. An Opportunistic Pathogen causes disease in a host that is physically impaired or debilitated. Normally the opportunist organism is harmless, but it takes advantage when the hosts defences are impaired, for example when the normal flora have been destroyed by antibiotics, or when the immune system has been suppressed by drug treatment or by other illnesses.

The relationships between the human host and most normal flora usually fall under the category of mutualist relationships. The benefit to the bacteria is that they have a place to survive and multiply. The benefits to the human host are as follows

The hosts ability to nourish itself is increased. The bacteria may produce vitamins (such as B and K), and may break down food stuffs that are normally indigestible by the host into components that can be digested.

The host is protected against infection by pathogenic organisms. This happens in several ways. Firstly, the normal flora occupies all of the available niches for bacteria, thus presenting the invading pathogen with the problem of finding somewhere to anchor itself. Secondly, the normal flora may outcompete the invader for the available food, thus starving the invader and preventing it from multiplying. Thirdly, some members of the normal flora produce antibacterial chemicals (bacteriocins) as a side product of their metabolism, thus generating a local antibiotic effect.

But not all normal floras are beneficial. If they remain in the site with which they are usually associated, the normal flora is usually beneficial. However, some members of the normal flora are also opportunistic pathogens, or are pathogenic if they turn up at a site with which they are not normally associated. Bacteroides bacteria, which normally reside in the intestines, may produce abscesses if they penetrate into deeper tissues via traumatic or surgical wounds. E.coli, a normal inhabitant of the gastrointestinal tract, is the most common cause of urinary tract infections.

Normal flora induces an immune response from the host. Antibodies to the normal flora exist in humab bodies, but at lower concentrations than would exist for pathogenic bacteria. They provide a "sparring partner" for bodies that keeps the immune systems in tune. However, the precise role that immune systems take in regulating the populations of the normal flora is not known. This experiment was done to identify types of the normal flora that exist at six different site of human body which are the skin, ear, nasal, mucous membrane, throat and rectal. Materials: 1. MacConkeys agar 2. Blood agar 3. Nutrient agar 4. SDA agar 5. Wire loop 6. Bunsen burner 7. Cotton swab 8. Grams stain 9. Slides 10. Microscope 11. Tongue depressor 12. Sterile saline 13. 70% alcohol 14. Soap 15. Toothpick 16. Oxidase reagent

17. Filter paper 18. Catalase reagent 19. Peptone water 20. MR/VP agar 21. TSI agar 22. Motility test medium 23. Citrate medium 24. Methyl red reagent 25. Voges-proskauer reagent 26. Kovacs reagent (Indole) 27. Dropper 28. TSI agar medium 29. Motility test medium Method: Day 1; 1. Six different agar plates were prepared for culturing human normal flora at six different places. 2. A cotton swab was used to swab inside nostril then it was swab on the blood agar. Then it was striking. 3. Another cotton swab was used to swab inside the ear lining then it was swab on the blood agar. Then it was striking. 4. A cotton swab was used to swab at the mucous membrane. Then it was swab on the SDA agar and it was striking.

5. A cotton swab and a tongue depressor were used to swab at the back of the throat. Then it was swab on the blood agar and was striking. 6. Cotton was deep into the sterile saline then it was used to swab at the rectal area. Then it was swab on the MacConkeys agar and striking. 7. A nutrient agar was divided into three regions. One region for unwashed finger to touch, one region for a finger that was washed with soap to touch and one region for a finger that was dipped into disinfectant or 70% alcohol to touch. 8. Then all the agar plates were incubates for 24 to 48 hours in the incubator at temperature of 37C. Day 2; 1. After 24 to 48 hours incubation, the cultured microbes were observed it culture characteristics and Gram stain were done. 2. The slides were then observed under microscope and Gram positive and negative was identified and differentiated. 3. Then, a few biochemical tests were run to observed it reactions. 4. For the Gram positive bacteria, three biochemical tests were run that were oxidase test, catalase test and coagulase test. For Gram positive chain, only oxidase and catalse tests that were run. 5. For Gram negative bacteria, biochemical test that run were IMViC tests, TSI tests and motility tests. 6. For all the tests, after striking and mixed, the entire mediums were incubated for 24 to 48 hours at 37C. 7. For agar plates that had no growth, they were reincubated. Day 3; 1. After 24 hours, the results of catalase tests, TSI tests and motility tests were observed and recorded.

2. For the indole test, MR test and VP test, a few procedures were done to obtain results. 3. For indole test, the cultured media were added with 5 drops of Kovacs reagent. The changes in the tube were observed and recorded. 4. For MR test, the cultured media were added with 5 drops of MR (methyl red) reagent. The changes were observed and results were recorded. 5. For VP test, the cultured media were added with 6 drops of VP 1 and 2 drops of VP 2. It was let mixed for 2 minutes. Then the changes were observed and results were recorded.

Result:

Ear

M. membrane

Nasal

Skin
Locations/ Tests Ear G+ (cocci) Catalase Coagulase Oxidase Indole MR VP Citrate TSI Motility
Positive Negative Negative Mucous membrane

Throat
Nasal G+ (cocci)
Positive Positive Negative -

Skin G- (cocci)
Negative Negative ? Negative K/K Negative

Throat G+ (cocci)
Positive Negative Negative -

G+ (cocci)
Negative Negative Negative -

G- (cocci)
Negative Negative ? Negative K/K Positive

G+ (cocci)
Positive Positive Negative -

Rectal: No growth (Reincubate) Discussion: From this experiment, the slide that was observed under the microscope and it showed presence of Gram positive cocci for ear culture, mucous membrane culture, and nasal culture. Meanwhile for skin culture and throat culture, both showed presence of both Gram positive cocci and Gram negative cocci. All the identified bacteria were then tested with biochemical tests. For the Gram positive cocci the tests that were run were oxidase, catalase and cougulase only. While for Gram negative cocci, the tests that were run were IMViC test, motility test and TSI test only. The results from these biochemical tests were listed in the result table above. For rectal culture, since there was no growth after 24 hours, it was reincubated for another 24 hours. The next day there was a little growth on the culture. The reason

for this problem may be due to the technique that was used when taken the sample was incorrect. The bacteria that were identified from this experiment for Gram positive are Staphylococcus aureus while bacteria of Gram negative are Escherichia coli. However there are many other species of normal flora that exist at those areas. The surface of the skin comprises several distinct environments. Areas such as the axilla (armpit), the perineum (groin) and the toe webs provide typically moister regions for bacterial growth. These "tropical forest" environments often harbor the largest diversity amongst the skin flora. Typical organisms include Staphylococcus aureus, Corynebacterium and some Gram-negative bacteria. The bulk of the human skin surface, however, is much drier and is predominantly inhabited by Staphylococcus epidermidis and Propionobacterium. Streptococci predominate in the oral cavity and nasopharyngeal regions but one can also find other anaerobes and species of Neisseria. Many potential pathogens may also be found in the nasopharynx of a healthy individual, providing a reservoir for infection of others. These pathogens include Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae.

Conclusion: As a conclusion, from this experiment there are millions of microorganisms that live on and in human body and some give good effects on human, some does not have effect on human and some may be dangerous to human. Human need to identify what are the normal floras and what are not so they can identify easily if there are any infection. Questions: 1. Explain what an immune compromised individual is?

- Immune compromised individual is individual that have impaired immunity such as those with AIDS, organ transplant, or bone marrow transplant, and people receiving chemotherapy or other immunosuppressive treatments. 2. What does normal flora mean? Normal flora is the assemblage of microorganisms that reside on the surface and in deep layers of skin, in the saliva and oral mucosa, and in the gastrointestinal tracts, including bacteria, fungi and archaea. These organisms are known to perform tasks that are useful for the human host. 3. Describe the three types of RBC hemolysis you observed or looked for. 4. Name two common diseases caused by Streptococcus sp. Two common disease caused by Streptococcus sp. are meningitis and bacteria pneumonia. 5. Can a healthy person have a throat containing a lot a beta-hemolytic strep? Explain. No, because beta-hemolytic strep is not part of the normal throat flora. Streptococcus pyogenes or Group A Strep is responsible for disease in the throat. 6. What is the relationship between the ability to lyse RBC and virulence? 7. What is the gram reaction of the Neisseria. Gram negative reaction.

8. Name two important diseases caused by Neisseria species. Two important disease caused by Neisseria species are gonorrhea and meningitis. 9. Describe how the nasopharynx strains are passed between hosts.

10. How does the oxidase test work and what does it detect?
-

A drop of oxidase was drop on litmus paper on a slide then a colony of culture bacteria were put on the litmus paper and the color change were observed. This test was used to detect presence of cytochrome-c from the bacteria. All of these bacteria use oxygen as the terminal electron acceptor in their respiration.

11. What is chocolate agar? Chocolate agar is a non-selective, enriched growth medium. It contains red blood cells. It is used for growing fastidious respiratory bacteria. 12. Explain why it makes evolutionary sense that staphylococci are tolerant to salt and drying? 13. What is the role of phenol red in the mannitol-salts medium? Phenol red is use as a pH indicator in the mannitol-salt medium.

14. Name four major diseases caused by S. aureus. Four major diseases cause by S. aureus are toxic shock syndrome, skin infections, pneumonia, and bacteremia. 15. Can you explain the differences in infection rate of S. aureus between the general public and hospital staff? [In public may be due to direct touch with infected people with S. aureus. Besides that the infection are because of pet like cat. Infection in hospital staff may due to the contaminated of the surgical or hospital instruments.] 16. Define the difference between selective and differential media and give examples of each. Selective media only grows certain types of microbes while inhibiting the growth of other types of microbes while differential media distinguishes between different types of bacteria based on some characteristic of the bacteria

that is growing on it. Examples of selective media are eosin methylene blue, blood agar, and Hektoen enteric agar. Examples of differential media are eosin methylene blue, MacConkeys agar, and Mannitol Salt agar.