Update, 08/04/30
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[菌数測定と増殖曲線]

Biochemical Determination of Bacterial Genus

INVIC

Prestudy

  1. What is enzyme ?
  2. How are glucose and lactose broken ?
  3. Why do you need so many kinds of culture media for determination of the bacteria ?
  4. What is the colors of the media and what porpose is it for ?

Aim:

Each student is supplied with two tubes, named Specimen R [red-capped] and Specimen B [blue-capped])of bacterial suspensions(Enterobacteriaceae)

The aims of this investigation are :

  1. To confirm the gram-negative rods and gram-positive cocci.
  2. To determine the Genus of the bacterial specimen supplied (Enterobacteriaceae) from the biochemical characters of the bacteria.

Instruments and Materials

  • Day 1
  • Day 2

    Preparation of media

    Day 1

    Media for determinative culture (team working, two each medium tube/student)

  • Prepare 20 tubes (per group, 2 strains/person x 8 persons/group + extra 4 tubes=20 tubes) of following media by the method as described.

    For about 20 tubes, make 60 or 100 ml of medium, each, in a clean either 100-ml or 200-ml flask; dissolve agar media by heating with the microwave; dispense small test tubes each with 3 or 5 ml of media with plastic dropper pipettes.

    Cover the tubes with plastic caps on as follows:

    Kliglerblue
    SIMwhite
    Lysine (LIM)green
    Simmon's Citratered
    VPred

    Install the tubes into tuberacks.

  • Autoclave for 15 min at 121 C.
  • Solidify the medium overnight at on the laboratory bench (Simmon's Citrate,slant; Kligler, half-slant; the others, deep stab agar).

    Day 4

  • Install tubes of solidified media into a tuberack.

    Procedures -Determinative culture-

      Day 1

    1. Inoculate the bacterial suspension into a tubes of Heat Infusion broth.
    2. Incubate the broth overnight at 37 C at standing posion.

      Day 2

    3. Inoculate the culture of gram-negative unknown bacteria into determinative media by stabbing and/or spreading on slants of media (see here to know how to inoculate to butt and streak into stab and slant media.
    4. Incubate overnight or more days at 37 C.

      Day 3 (or later)

    5. Observe the changes of media.

      For VP reaction in VP medium and Indol reaction in SIM and Lysine, the reaction reagents are used.

      Interpretation of Kligler slant/butt reactions
      Features Marks Indication
      A red slant with yellow buttK/AOnly glucose was fermented
      A yellow slant with yellow buttA/ALactose were fermented in addition to glucose.
      A red slant with red buttK/KNone of the sugars were fermented and only the peptone supported growth.K/K tubes will only have growth on the slant.
      Bubbles or cracksG Gas production
      A black butt or blackening along the stabSH2S production.
      A:Acid ; K:Alkali, If the slant is red with H2S, bud is yellow with gas production, it should be noted as "KS/AG".

    6. Estimate the species through the following diagram of biochemical characterizations of Enterobacteriaceae.