This study was carried out among selected pigeon farm located within Abakaliki metropolis. Abakaliki is the capital of Ebonyi State, South-East Nigeria. It is the largest city in this State and it is inhabited by majorly Igbo speaking tribe. The occupation of the indigenes of Ebonyi State is mainly farming. This region is characterized by an annual rainfall of 2500mm and it is located on coordinates 6˚20ˈN 8˚06ˈE. The selected poultry farm is located within Abakaliki town.
- Bijuo bottles, Microscope, Wire loop, Bunsen burner, Petri dishes, Sterile Swab Sticks, Test tubes, weighing balance, Autoclave, Conical flask, Slides.
- MacConkey agar, Eosin methylene blue agar, Tryptone Soy broth, Simmon’s citrate agar,Nutrient broth.
- Crystal violet, 70% Acid Alcohol, Lugols iodine, dilute Carbol Fuchsin, Kovac’s reagent.
All media (Blood agar, MacConkey agar and Eosin methylene blue agar) used in this study were aseptically prepared and used according to the manufacturer’s (Oxoid company, United Kingdom) instruction.
A total of 50 samples of Pigeons were collected.the samples were aseptically collected from different pigeon cloaca with the use of sterile swab stick. Each Pigeon from which a sample was collected was marked appropriately to avoid repetition.
Inoculation of samples
The cloacal swab sample was used to inoculate a prepared sterile nutrient broth contained in a test tubes. The inoculated broths were incubated at 37C for 18 -24 hrs.
Purification of isolates from Pigeon samples
A sterile wire loop was used to pick a loop full from the incubated broth and was inoculated on sterile MacConkey agar plates for differentiation. The plates were inoculated at37C for 18 -24 hrs. The suspected colonies were subcultured on EMB agar .they were also incubated at 37C for 18 -24 hrs. The colonies were then transferred to nutrient agar slants for further use.
Characterization of isolates
E.coli and Klebsiella spp were identified and characterized by their colony morphology on Macconkey and EMB agar.Pure colonies were transferred to a 5ml nutrient agar slant and stored in the refrigerator for further use. Gram staining reaction and other biochemical test were also carried out to confirm the isolates.(Cheesbrough, 2002).
Gram staining is a differential stain and is the most widely used staining procedure in bacteriology. It aids in differentiating bacteria into two groups; gram positive and gram negative. The age of the culture is very important in this procedure. A smear of the organism to be investigated was formed on a grease-free, dry glass slide using a sterile wire loop,
The smear was allowed to air dry and then heat-fixed by passing it through Bunsen burner flame. The heat-fixed smears were stained for 30-60 seconds with crystal violet (primary stain) and the excess were washed with slowly running tap water. Lugol’s iodine was later added for 30-60seconds serving as a mordant. The excess iodine was rinsed off with slowly running tap water. The smears were then rapidly decolorized with acetone – alcohol for few seconds and immediately washed with clean water.
Finally the smear were counter stained with safframine red (secondary stain) for about 30- 60 seconds , after which they were rinsed with water and allowed to dry and the slides was viewed under the microscope using x100 oil immersion lens. Gram negative organisms appeared pink while gram positive organisms appeared purple. (Cheesebrough,2006).
Biochemical Identification and Characterization of Isolates
Biochemical tests employed were indole test, methyl red test, Voges-Proskauer test, citrate utilization and Motility test . For biochemical tests, standard procedures, according to Ochei and Kolhatkar, (2000), were used.
Indole Production Test
The test depends on the ability of a bacterial isolate to breakdown the enzyme, tryptophase to produce indole. Loopful of the isolate was aseptically inoculated into a test tubes containing sterile tryptone water and incubated at 37oC for 24 hours. After incubation, 0.5ml of Kovac’s indole reagent was added into the culture tubes. The tubes were shaked and left to stand for about 10 seconds . A reddish ring at the upper layer indicated indole production while no reddish ring indicates no indole production, (Cheesebrough,2006).
Methyl Red (MR) Test
This detects the ability of an organism to produce and maintain stable acid end products from glucose fermentation. Some bacteria produce large amounts of acids from glucose fermentation, that they overcome the buffering actions of the system. Methyl Red is an indicator which remains red in colour at a pH of 4.4 or less.
The test organism was inoculated into Bijou bottles containing 3 ml of sterile glucose phosphate broth and incubated at 37oc for 24 hours. Five drops of methyl red agent was added and examined. Development of red colour indicated a positive methyl red test while a yellow colour indicated Negative methyl red test.
Voges-Proskauer (VP) Test
Many bacteria ferment carbohydrates with the production of acetoin. In the presence of potassium hydroxide and atmospheric oxygen, acetoin is converted to diaetyl and alphanaphthol which serves as a catalyst to form a red complex (Ochei and Kolhatkar, 2000). The test organism was inoculated in glucose phosphate broth and incubated at 37oC for 48 hours. 1ml of potassium hydroxide and 3ml of 5% solution of alphanaphthol in absolute alcohol were added. A positive reaction is indicated by the development of pink colour in 2-3 minutes.
Citrate Utilization Test
Slants of Simmon’s Citrate agar were prepared in test tubes according to the Manufacturer’s instructions. Using a sterile straight wire, the slope was first streaked with a saline suspension of the test organism and the butt was then stabbed. The test tubes were incubated at 37˚C for 24 hours. A change in colour of the citrate slants from green to bright blue indicates that the test is positive while retention of green colour indicates a negative test, (Ochei and Kolhatkar, 2000).
Label the tubes of semisolid (or SIM) media with the names of the organisms.Flame and cool the inoculating needle, and insert it into the culture after flaming the neck of the tube.Remove the cap from the tube of medium, flame the neck, and stab it 2⁄3 of the way down to the bottom. Flame the neck of the tube again before returning the cap to the tube.Incubate the tubes at room temperature for 24 to 48 hours.Examine the SIM cultures for the presence or absence of a black precipitate along the line of the stab inoculation. A black precipitate of FeS indicates the presence of H2S.If desired, one can also test for indole production by adding 5 drops of Kovacs’ reagent to the SIM cultures and looking for the development of a red color at the top of the deeps.