Lactobacillus acidophilus is a gram-positive bacterium commonly used in yogurt p

Question

Lactobacillus acidophilus is a gram-positive bacterium commonly used in yogurt production. It is an obligate homofermentative bacterium, which means that it is only capable of carrying out fermentation metabolism to produce lactic acid. It is also an aerotolerant anaerobe, which means it is able to tolerate atmospheric oxygen concentrations but does not use oxygen in its metabolic processes. Describe the results you would expect to obtain if you tested this bacterium using the oxidase test, nitrate reduction test, EMB agar test, and blood agar test. For each, explain the reason you would expect to observe that result.

Explanation / Answer

Lactobacillus acidophilus is a gram positive aerotolerant bacteria that can tolerate oxygen but does not utilize oxygen in metabolism.

Oxidase test: Oxidase test is used for identification of bacteria that produce cytochorme c oxidase, a haeme containing protein. Bacteria may not be strictly aerobic even if they can use oxygen as a terminal electron acceptor in respiration. The positive oxidase test indicates that the organism maybe aerobic, anaerobic or facultative aerobic as it only detect the enzyme. Cytochrome c oxidase is able to convert tetramethyl-p-phenylenediamine to indophenols, which gives a purple color. Oxidase negative bacteria utilize other oxidases to respire. The bacterial colony is picked with a wooden or platinum loop and smeared on a filter paper soaked with the tetramethyl-p-phenylenediamine. If a purple (blue) color results, it will be oxidase positive.

Lactobacillus acidophilus is a homofermentative bacterium and can convert glucose entirely into lactic acid. They are oxidase negative and hence do not have haeme containing cytochrome oxidase. Instead, they have NADH oxidase. NADH oxidase causes reduction of molecular oxygen forming superoxide ion, which reacts with H+ and forms hydrogen peroxide. Hydrogen peroxide is broken down to water by NADH peroxidase.

Test results: negative

Nitrate Reduction test: Nitrate broth is used to determine whether an organism can convert nitrate to nitrite to form nitrous acid. Addition of sulfanilic acid and -naphthylamine is performed. If nitrous acid is formed, it will react with sulfanilic acid to form diazotized sulfanilic acid, which reacts with -naphthylamine to form a red colored compounds. A positive test is formation of red colour. Lactobacillus acidophilus is nitrate negative and cannot reduce nitrate to nitrite. Hence, it is negative result for nitrate as it cannot break down nitrate to nitrite due to lack of nitrate reductase. However, it can break down nitrite to nitric oxide. Lactic acid produced by this bacteria leads to medium acidification. This acidic pH in turn generates nitric oxide by non-enzymatic nitrite reduction.

Test results: negative

EMB Agar test: Eosin methylene blue agar has eosin and methylene blue that allows the growth of gram negative bacteria but prevents gram negative bacteria from growing. Gram negative bacteria that ferment lactose give purple colonies on this agar due to fermentation of lactose. Lactic acids produced will lower the pH of the medium, to give purple color. These organisms will give purple colonies with or without green metallic sheen. Lactose non fermenters will be colourless or light green. Although lactobacillus acidophilus can ferment lactose, it is gram positive. Hence, it will be inhibited by eosin and methylene blue added.

Test results: no growth

Blood agar test: Blood agar contains 5% sheep blood and is used to cultivate fastidious organism. Bacteria that produce hemolysins that lyse red blood cells and degrade hemoglobin. Beta-hemolysin breaks RBCs and hemoglobin completely, leaving a clear zone around the colony (beta hemolysis). Alpha-hemolysin causes partially breaks down the red blood cells to form a greenish color (alpha hemolysis) due to presence of biliverdin (a byproduct of haemoglobin). If there is no hemolysin produced no clearing will occur (gamma hemolysis). Lactobacillus acidophilus is alpha haemolytic showing a green coloration around their colonies on blood agar.

Test results: Alpha haemolysis (green coloured colonies)

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