BASIC TESTS FOR IDENTIFICATION OF BACTERIA
A. SMEAR PREPARATION
The preparation of a smear is required for many laboratory procedures, including the Gram-Stain. The purpose of making a smear is to fix the bacteria onto the slide and to prevent the sample from being lost during a staining procedure. A smear can be prepared from a solid or broth medium. Below are guidelines for preparing a smear for a Gram-stain.
• Wear gloves
• Avoid aerosols
• Care must be taken when heat fixing slide
• Glass slide
• Sterile normal saline
i. Add one drop of sterile normal saline or distilled water on a clean glass slide
ii. With a sterile loop add 1 colony of bacterial growth to the water,…show more content… Observe for the evolution of gas.
Result Evolution of a gas (Oxygen) causes bubbles – Positive No evolution of gas – Negative
Cytochrome oxidase is an enzyme found in some bacteria that transfers electrons to Oxygen, the final electron acceptor in some electron transport chains. Thus, the enzyme oxidises reduced cytochrome C to make this transfer of energy. Presence of cytochrome oxidase can be detected through the use of Oxidase Discs, which acts as an electron donator to cytochrome oxidase. If the bacteria oxidises the disc, (remove electron) the disc will turn purple.
i. Make a light suspension of oxidase reagent (1% N-tetramethyl-P-Phenyldiamine dihydrochloride) in sterile distilled water. ii. Transfer few drops on a clean white filter paper on a glass slide. iii. Add on the test organism with an applicator stick (do not use aluminium loop). iv. Observe for colour change.
Results Purple colour – Positive No colour change – Negative INDOLE TEST
Indole is a component of the amino acid tryptophan. Some bacteria have the ability to break down tryptophan for nutritutional needs using the enzyme tryptophanase. When tryptophan is broken down, the presence of indole can be detected through the use of Kovac’s reagent. Kovac’s reagent reacts with indole and produces a red colour on the surface of the test…show more content… Therefore all probable Streptococcus pneumoniae that are resistant to optochin must be confirmed serologically or by PCR.
This helps to differentiate Streptococcus pneumoniae, which is soluble in bile and bile salts from other alpha haemolytic Streptococci e.g. Viridian Streptococci which are in soluble.
i. Emulsify several colonies of suspected Streptococcus pneumoniae in 2ml of normal saline to give a turbid suspension. ii. Divide the suspension between two tubes – one labelled ‘test’ and the other ‘control’. iii. To the ‘test’, add two drops of Sodium deoxycholate reagent and mix. iv. To the control tube, add two drops of distilled water and mix.
v. Incubate both tubes at 37oC for 15mins vi. Look for clearing of turbidity.
Clearing of turbidity – probably Streptococcus pneumoniae
No clearing of turbidity - probably not Streptococcus pneumoniae
To make Sodium Deoxycholate reagent: -
Sodium Deoxycholate – 2g
Sodium chloride 8.5g/l – 20mls
Autoclave at 121oC for 15mins, label and store in