Question

1. When you try to transfer bacteria from one plate to another, why is it advisable to pick one single colony?

2. Explain how you would measure bacterial growth in agar plate and in broth culture (test tube). Polymerase chain reaction is not the answer

3. Catalase and cytochrome c oxidase are two enzymes that are used to type or identify bacteria. What type(s) of bacteria would test positive for these two enzymes?

Answer 1

1. While transfering bacteria from one plate to another it is advisable to pick one single colony inorder to achieve a pure culture. This is because each colony represents the descendants of a single bacterial cell. So all the cells in that colony are clones. (clones are cluster of cells arising from a single bacterium by asexual reproduction)

2. bacterial growth in agar plate or in broth culture can be calculated by using serial dilution technique.

1cm3 or 1ml of each of the diluted sample( just see the image above). are individually streaked in to sterile agar plates, and incubated at 25 degree celcius fro 2 days for the bacteria to grow. After all the streaks have been allowed to grow, the dilution at which the colonies are distinctly and seperatly identified is noted down. Let it be the 1/1000 dilution from the example above. If the dilution is insufficient then colonies will merge, referred to as ‘clumping’, and counting is inaccurate which is noted in 1/10 and the 1/100 dilution. The distinct and separate colonies of bacteria from the agar plate that has been chosen are counted with the assumption being made that each colony has arisen from a single cell, which has divided asexually, from the original culture medium. To find the total viable cell count the number of colonies in the selected plate and multiply by the appropriate dilution factor. So in the example above this would be:

140 colonies counted on the 1/1000 dilution agar plate
So 140 x 1000 = 1.4 x 10 to the power 5 bacteria in the original sample.

3) catalyse and cytochrome coxidase are the enzymes present in specific group of bacterias.

Catalase test :

principle: certain bacteria have en enzyme catalase which acts on hydrogen peroxide to release oxygen.

H2O2 in presence of catalase gives H2O (water) and nascent O2 (oxygen)

procedure:

the bacteria colony is picked from the culture medium. It is mixed with a drop of H2O2 on a clean glass slide. A positive catalase reaction produces gas bubbles (nascent oxygen)

Interpretation :

positive test - iimediate buubling which is easily seen (oxygen formed)

negative test - no buubling (no nascent oxygen is formed)

Positive and negative catalyse bacteria

Catalase positive bacteria :

all members of the enterobacteriaceae except shigella dysentriae type 1, staphylococcus, micrococcus

catalase negative bacteria : shigella dysentriae type 1, streptoccus

catalase test is primarily used to diiferentiate between genera staphylococcus from streptococcus both of which are gram positive bacteria

OXIDASE TEST

Principle :

to determine the presence of an enzyme cytochrome oxidase which catalyses the oxidation of thereduced cytochrome by molecular oxygen

procedure:

freshly prepared solution of 1 % tetramethyl paraphenylene diamine dihydochloride (oxidase reagent) is used. There are various methods to used perform this test.

i. a filter paper strip soaked in the oxidase reagent is smeared with test organism. A positive reaction turns in deep purple color (indophenol is formed) within 10 second.

ii.another method is to pour oxidase reagent on the surface of colonies, the colonies become purple within 10-30 minutes. this technique is used to pick up Neisseria colonies from mixed growth on the culture media.

Interpretation :

positive - deep purple color within 10 seconds

negative - no color change

Positive and negative oxidase bacteria

oxidase positive bacteria are : pseudomonas species, vibrio species and neisseria species

oxidase negative : all the members of eneterobacteriaceae

The oxidase positive bacteria are aerobic but not strict aerobes which uses oxygen as terminal electron acceptor in respiration. This enyme is present on the membrane of the bacteria. Oxidase helps the bacteria to accept the electron from the external environment.