Question

Explain the chemical principle of the three types of medias used to isolate C. diptheriae from a throat swab? Must include contents, function of chemical in the media, and how media is interpreted based on indicators) List the general type with each media (Selective, Differential etc.)

Answer 1

Diagnostic tests used to confirm infection combine isolation of C diphtheriae on cultures with toxigenicity testing. In all patients in whom diphtheria is suspected and in their close contacts, obtain specimens from the nose and throat (ie, nasopharyngeal and pharyngeal swab) for culture.

several types of medias are used to isolate the C.diptheriae from the throat swab. the main three medias are  Loeffler agar, Mueller-Miller tellurite agar, or Tinsdale tellurite agar

Loeffler agar medium

This media contains horse serum, beef extract, dextrose and proteose peptones which together supply the complex nitrogenous substances and nutrients necessary to support the growth of C. diphtheriae . Sodium chloride is added to supply essential ions. This media enhances the formation of microchromatic granules and it demonstrates the cellular morphology of the bacteria. hence the presence can be detected.

prior to enoculation, the media should be bring to room temperature. inoculate the swab in a fishtail motion into the media and incubate aerobically at 35ºC. for up to 4 days. observe everyday for the colonization of bacteria. check for the presence of metachromatic granules and appearance suggestive of chinese-letter formation of cells.

Mueller-Miller tellurite agar

This media is used for the isolation, cultivation and differentiation C. diphtheriae. The chemical composition consists of Casein acid hydrolysate, Casein powder,Potassium dihydrogen phosphate,Magnesium sulphate. heptahydrate,L-Tryptophan,Agar, and Final pH ( at 25°C).

A 45.45 grams of dehydrated media is suspended in 975 ml distilled water. The medium is gently heated and boiled till the medium is dissolved completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool quickly to 50°C and aseptically add 25 ml Mueller Tellurite Serum (FD100). Mix thoroughly to distribute into sterile Petri plates. Allow the surface of the plates to dry by partially removing the covers during solidification.

interpretation: C. diphtheriae is gram-positive, facultative anaerobic, non-motile bacteria. The potassium tellurate in the media inhibits the growth of most of the microorganisms in the respiratory tract except diphtheriae. The serum used in medium enhances granule formulation. The factors in the media which provides nitrogenous compounds for the growth area Casein acid hydrolysate and L-tryptophan . The magnesium sulphate element which supplies essential ions required by the organisms. . grayish black colonies surrounded by dark brown halo due to H2S production are formed by the bacteria. hence it can be detected.

.Tinsdale tellurite agar

This is also another media used for the isolation of C.diphtheirae. The chemical composition includes Peptic digest of animal tissue, Sodium chloride, L-Cystine, Sodium thiosulphate, Agar, and Final pH ( at 25°C).

A 40.67 grams of media is suspended in 1000 ml distilled water. The medium is heated to boiling until it dissolve completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 50°C and aseptically add Diphtheria Virulence Supplement (FD073, Part A and Part B). Mix well and pour into sterile Petri plates.

Corynebacteria are gram-positive, non-sporulating, non-motile rods. they are seen as rod shaped , frequently banded or beaded with irregularly stained granules.

The growth of other organisms and all gram negative bacterial growth are inhibited by the Potassium tellurite from the supplement. The peptic compound is the nitrogenous supplying agent. C. diphtheriae forms grayish black colonies surrounded by a dark brown halo while diphtheroids commonly found in the upper respiratory tract do not form such colonies.