We were provided with number coded 24- hour broth culture containing a mixture of any two bacteria (gram +ve or gram –ve), 1 EMB plate, 1 blood agar plate, several inoculating loops and TSA slants. Other materials (mediums and reagents) required were requested from the laboratory technician depending on the type of tests that were to be carried out to facilitate in identifying the unknown bacteria.
There were several tests to be carried out to identify the unknown bacteria. On receiving the less than 24 hours broth culture which contained two types of bacteria (G +ve and G- ve), I prepared a stock culture for culture purification. Labelled the bottom of blood agar plate (nonselective medium) with my identification codes, then chose one of the unknown culture, opened the petri dish carefully without placing the top of dish down on the bench. I used the inoculating loop to gently swipe across an area of the bacteria to transfer from the dish to the loop and gently made three successive streaks of the bacteria onto the agar surface of the labelled plate. Incubated the plate at 35o C for almost a week, I kept checking it regularly until single colonies had grown. Then I made a subculture of a single colony on TSA slant, incubated it at 35o C after good growth, I removed from the incubator and stored at room temperature.
After acquiring the individual colonies, I performed a gram stain test using EMB (eosin Methylene blue) and lactose agar plate. Collected bacterial colony from and streaked on the EMB plate and waited for some days to observe the result. The unknown bacteria was Gram negative. With G –ve result I had to carry more tests: Oxidase test, Lactose fermentation test, Indole test, Bile Esculin test, Motility test, Methyl Red -Voges Proskauer test, and Lysine decarboxylase test.
Oxidase test: Obtained a liquid culture stock of my gram