The study was conducted from November 2009 through March, 2010 which corresponds to the dry season when droplet infections thrive most due to dry weather and when the dust in Abakaliki, the capital city of Ebonyi state reaches its peak. This area lies approximately between longitude 8061611 E and latitude 602212611 N and is located on the lower belt of Niger. Ebonyi state according to 2006, census has a population of 2,173,501 and Abakaliki the state capital approximately 141,438. The climate is tropical and the vegetation characteristic is predominantly the semi- tropical rain forest. There are two distinct seasons, the wet and dry seasons. The former occurs between April and October, while the latter takes place from November to March. During the dry season the area is very dusty due to many Quarry Industries and stone Mines within and outside the metropolis.
The city has three major hospitals for treatment of infectious diseases and other kinds of ailments. One of the Hospitals, Mile Four Hospital is a mission hospital and is one of the biggest Leprosy and Tuberculosis centres in the south east Nigeria. The daily out-patients in the out patient departments (OPD) these Hospitals formed our study population. Our respondents are both male and female patients above ten yeatrs of age.
MATERIALS
REAGENTS: The reagents used include, carbol fuchsin strain, 3% v/v acid alcohol, methylene blue, crystal violet stain, lugol’s iodine, acetone alcohol, neutral red stain.
EQUIPMENT: Microscope (Nikon) Refrigerator (Thermo cool Nig.), sputum container, glass wares, forceps, slide box, staining bridge, Bunsen burner, Petri-dishes, and conical flask (England UK).
METHODS:
SAMPLE COLLECTION: One thousand five hundred (1,500) sputum samples were collected from respondents over a period of five months, from November to March 2010.
Five Hundred samples each were collected from the out patients departments (OPD) of the three hospitals, Ebonyi State University Teaching hospital, Federal medical centre and Mile Four Hospital.
After an informed consent, sputum sample is collected randomly from the respondent into a clean wide mouthed sample bottle on first contact. Sample bottle is given to every third patient who is within our age range ie above ten years.
The sputum is taken to the laboratory for examination and analysis.
Macroscopic examination of sputum: The sputum samples were observed and the volume, appearance, colour especially (presence) of blood) and consistency were recorded.
Centrifugation of sputum samples: About 2 -4ml of sputum sample is transferred to a screw cap universal bottle of 20ml capacity. 2-4ml of concentrated sodium hydrochloride is added and mixed throughout. It was left at room temperature (350C) for 10-15 minutes, shaken at intervals to break down the mucus in the sputum. A 4.8ml volume of distilled water was added and well centrifuged at 500 rpm for 15 minutes and the supernatant fluid was discarded and the sediment collected for further analysis.
SCREENING FOR M. TUBERCULOSIS USING ACID FAST METHOD (ZIEHL-NEELSEN STAIN)
A sterile loop was used to transfer and spread purulent and caseous material on a microscopic slide. It was allowed to air dry and the slide washed with 70% acid alcohol. The slide is then covered with carbol fuchsin stain and heated until vapour began to rise. The heated stain was allowed to remain on the slide for 5 minute after which the sleeve was washed off with clean water. The smear was then covered with 3% v/v acid alcohol for 5 minutes and the slides was washed off again with clean water. The smear was then covered with methylene blue for 1-2 minutes and washed off with clean water and placed in a draining rack to air dry the smear. This was examined microscopically with oil immersion objective lens at x 100.
Biochemical test
Catalase test
2- 3ml of hydrogen peroxide solution was poured into a test tube and a piece of sterilized swab stick was used to remove several colonies of the test organism. This was immersed in the hydrogen peroxide solution and was observed for the production of bubbles within 1 minute.
Niacin accumulation test
1ml of sterile distilled water was added to the slant of I.J. culture medium. The culture was placed in a slanting position so that the fluid covers the mycobacterial growth. 0.5ml of the fluid is removed and placed in a screw-capped test tube. 0.5ml each of 4% alcoholic aniline and 10% aqueous cymogen bromide solutions was added. This was observed for few minutes for Niacin extraction in which case a yellow color would appear.
SCREENING FOR M. TUBERCULOSIS USING ACID FAST METHOD (ZIEHL-NEELSEN STAIN)
A sterile loop was used to transfer and spread purulent and caseous material on a microscopic slide. It was allowed to air dry and the slide washed with 70% acid alcohol. The slide is then covered with carbol fuchsin stain and heated until vapour began to rise. The heated stain was allowed to remain on the slide for 5 minute after which the sleeve was washed off with clean water. The smear was then covered with 3% v/v acid alcohol for 5 minutes and the slides was washed off again with clean water. The smear was then covered with methylene blue for 1-2 minutes and washed off with clean water and placed in a draining rack to air dry the smear. This was examined microscopically with oil immersion objective lens at x 100.
Biochemical test
Catalase test
2- 3ml of hydrogen peroxide solution was poured into a test tube and a piece of sterilized swab stick was used to remove several colonies of the test organism. This was immersed in the hydrogen peroxide solution and was observed for the production of bubbles within 1 minute.
Niacin accumulation test
1ml of sterile distilled water was added to the slant of I.J. culture medium. The culture was placed in a slanting position so that the fluid covers the mycobacterial growth. 0.5ml of the fluid is removed and placed in a screw-capped test tube. 0.5ml each of 4% alcoholic aniline and 10% aqueous cymogen bromide solutions was added. This was observed for few minutes for Niacin extraction in which case a yellow color would appear.
Neutral red test
5ml of ethyl alcohol was placed in screw – capped bottle and inoculated with 1-2 colonies from I.J. medium. The bottle is incubated at 370C for an hour. Supernatant alcohol is carefully removed with a pipette and 5ml of distilled water and 0.2ml of 0.05% aqueous solution of neutral red are added. The fluid was rendered alkaline by N/100 NaOH drop by drop till the colour is just amber. The bottle is further incubated in water bath at 370C for another one hour with frequent shaking. The colonies suspended in the amber fluid stained pink or red in positive cases.
5ml of ethyl alcohol was placed in screw – capped bottle and inoculated with 1-2 colonies from I.J. medium. The bottle is incubated at 370C for an hour. Supernatant alcohol is carefully removed with a pipette and 5ml of distilled water and 0.2ml of 0.05% aqueous solution of neutral red are added. The fluid was rendered alkaline by N/100 NaOH drop by drop till the colour is just amber. The bottle is further incubated in water bath at 370C for another one hour with frequent shaking. The colonies suspended in the amber fluid stained pink or red in positive cases.