Bailey and scott's diagnostic microbiology

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Sputum diagnosis in case of a cystic fibrosis patient may cause Pseudomonas species.

In healthy human body there is instinctive flow of mucus in both lungs. Mucus is very important thin natural liquid to protect the lungs from many foreign particles like bacteria. It also verify many harmful particles like dust and pollen which may cause severe infection of lungs. But in case those foreign objects are able to reach the lungs mucus then become as a form of cough. The patient who is suffering from cystic fibrosis mucus of his lungs is thick, sticky and greenish in color. Cystic fibrosis is a genetic disorder and it affects mostly the lungs, digestive system and also sex organs. It is characterized by abnormally thick mucus secretions in both lungs and by pancreatic enzyme insufficiency. Abnormal mucus secretion clogs the lungs which may cause breathing problems and it makes easy for bacteria to grow. This is responsible for some severe lung infections and lung damage.

Mutation in CFTR (CF transmembrane conductance regulator gene) gene is responsible for cystic fibrosis. Gene mutation of CFTR broken apart the activity of the chloride channels and stop them from regulating the flow of chloride ions and water across cell membranes. As a result, cells that lines the entrance of the lungs, pancreas, and other organs yield mucus that is very thick and sticky. This mucus blocks the airways and glands and causing the indicative symptoms and signs of CF. Sometimes these infections develop ineradicable coughing, heavy breathing problem, and inflammation. Increased thick and sticky mucus and infections arise permanent injury of the bronchi, including the establishment of some scar tissue (fibrosis) and cysts in the lungs. This serious situation oh the bronchi are generally known as bronchiectasis.

In case of the patient Benjamin (14years old) suffering from increased cough with thick green sputum, breathlessness, lethargy, weightlessness, rhonchi and crepitations throughout both lungs. He was diagnosed with CF in his early childhood and his elder brother has also CF. It is already proved that cystic fibrosis is one of the most common autosomal recessive conditions that means in each cell both copies of the gene have mutations. With an autosomal recessive condition both father and mother of a child carry onecopy of the mutated gene, but they never show indications and symptoms of the circumstances.

In the case study of the patient named Benjamin (14years) it is necessary to detect the main pathogen (bacteria) which is responsible for this chronic disease and to disclose the bacteria have to take sputum from Benjamin for pure culture of the bacteria. In order that he could have medication for prevention.

Materials used:

reagents for gram staining, those are primary stain- methyl violet or crystal violet, counter stain- neutral red or safranin, decoloriser- alcohol, mordant- iodine solution. To carry out the diagnostic test some agar plates are required for pure culture and they are, blood agar, nutrient agar, nutrient broth, MAC agar, and certified agar, MH agar plate for antibiotic sensitivity test, antibiotic strip, and apparatus for API20E test.


Staining: Gram staining is important to detect the bacteria even if it is gram positive or gram negative. Before staining some bacteria from culture was sweeped on a slide. After that crystal violet was added on it for a minute, then was cleaned up with water. All cells became purple in color. In the second step iodine reagent was included and have left for one minute. Again was cleaned with water and the cells kept on purple colored. After that alcohol was added and then was washed up by water for the third time. In this step gram positive and gram negative bacteria was indentified separately. In the final step safranin was included to give color to gram negative cells. Then the slide was washed by using water and was left to dry in air before watching under the microscope.

Plating for culture: Pure culture is a culture of single cell organism without any contaminants. Microbial culture is a process of multiplying the organisms by letting them grow in appropriate culture media plate under controlled laboratory conditions. For this blood agar, nutrient agar, MAC agar, nutrient broth, MH agar plates are taken and have left them for a week for perfect bacterial growth. To observe any haemolysis was occurred or not blood agar plate had been used. Nutrient agar and nutrient broth was taken for culture of the sample and MH agar plate was used for antibiotic sensitivity test of the sample.

Catalyze and oxidize test: After plating and making the culture plates catalyze and oxidize test carried out on the plate of pure culture to justify either it is gram negative or gram positive. Antibiotic resistance test was done on MH agar plate. There were few antibiotics had been used for that test, AP=ampicillin, GM=gentamicin, PY=carbenicillin, NA=nalidixicacid, NI=nitrofurantoin, SM=sulphamethizole, T=tetracycline, TS=cotrimoxozole were those antibiotics. After that certified media plate was arranged and was left for one week to achieve the appropriate result. After the oxidize and catalyze test cetrimmide and MSH media plates were applied to check out the sensitivity of the sample bacteria.

API 20 E test: API 20E test is basically an identification testing system for bacteria. This test is achieved for its diagnosis to prove perfectly the bacteria either gram negative or gram positive. In this test there was a plastic strip of 20 of different downsized tests tubes. Each of them consists of individual reagents and the determination of the metabolic capabilities was controlled by those reagents. Finally the identification process was finished and the enteric bacteria were from the family Enterobacteriaceae.


Following results were found during the experiment.

Staining: During the staining method it was proved that the microbe was rod shaped and gram negative bacteria.

Agar plates: Beta hemolytic had occurred in blood agar plate. As the previous staining method proved that the bacteria are gram negative, accordingly the bacteria must be an enteric bacteria. MAC agar plate changed its color to orange and this displays Pseudomonas species was present in the culture media. Certified agar media is most excellent culture media for Pseudomonas species and there were striking growth has been seen in this agar plate. Positive bacterial growth is noticed. So that, the bacteria may be Pseudomonas.

Catalyze and oxidize test: the results were positive in both catalyze and oxidize tests. In case of only gram negative bacteria oxidize test always give the positive result.

Antibiotic resistant test: For this test antibiotic strip was used and there was a zone of inhibition only for gentamicine (10 µg). The diameter of the zone of inhibition was measured.

API 20 E test: This final identification test affirms that large number of Pseudomonas aeruginosa was present in the sputum. All of the diagnosis before this test also indicated the existence of Pseudomonas species, but only the API 20E test confirmed that the bacteria which are mainly responsible for cystic fibrosis are Pseudomonas aeruginosa.


The diagnosis process was quite favorable in this particular case study and the whole experiment indicated the existence of Pseudomonas sp. This species is generally gram negative rod shaped aerobic and motile. During the staining procedure it was proved that the culture of the bacteria is gram negative. Generally beta hemolysis occurred in case of gram positive bacteria, in this case beta hemolysis takes place in blood agar, it can possible in case of gram negative enteric bacteria also. Again the result of oxidize was positive and this result is another evidence of the presence of gram negative bacteria in the sample. From the outcome of the antibiotic resistance test it was proved that only gentamicine is successful to resist the gram negative bacteria which are responsible for cystic fibrosis. Finally the API20E test approved the presence of Pseudomonas aeruginosa (67.1%) in the sputum of Benjamin.


The sputum of the patient suffering cystic fibrosis has traces of Pseudomonas aeruginosa. But in case of the specific sputum which was used in the whole experiment might have some contamination. So that the sputum contains some other species of pseudomonas. For this reason there is a confusion to deliver the report to the clinician to give medication to the patient having cystic fibrosis. The entire experiment analyzed that the patient who is suffering cystic fibrosis produces Pseudomonas aeruginosa, during the culture of their sputum.


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