7-3 Protocol for inoculation of medium

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Below is listed the typical classroom protocol. Read through it to get an idea of the steps necessary to perform the tests.

Materials

3 of each of the following medium per student:

Glucose Fermentation Broth (5 ml/tube)

Lactose Fermentation Broth (5 ml/tube)

Tryptone Broth (5 ml/tube)

Starch Agar Plates

Heart Infusion Agar (HIA) plates

Motility Agar Medium

Nitrate Broth

Cultures:

Escherichia coliEnterococcus faecalis
Klebsiella planticolaLactobacillus plantarum
Pseudomonas fluorescensBacillus cereus
Micrococcus luteusBacillus subtilis
Staphylococcus epidermidisBacillus polymyxa
Chromobacterium violaceumSerratia marcescens

The class will work in groups of 3 with each student receiving 3 cultures to investigate. Each student will be responsible for observing each test on every organism! It is usually most convenient to record data on the characteristics of microorganisms in tabular form.

  1. Perform a Gram stain on each of the pure cultures. Note the cell morphology and Gram reaction and be sure to examine the Gram stains prepared by other members in your group.
  2. Inoculate both of the fermentation broths with the cultures. Avoid shaking the tubes since this may trap an air bubble in the Durham tube, confusing the results.
  3. Streak a single line of each culture onto the middle of a plate of starch agar. Do not streak for isolated colonies.
  4. Streak each culture onto a plate of HIA for isolated colonies.
  5. Inoculate each culture into a tube of tryptone broth to test for the production of indole from tryptophan. Tryptone is a peptone that contains high concentrations of tryptophan.
  6. Nitrate reduction. Inoculate a tube of Nitrate Broth.
  7. Incubate all tubes and plates at 30°C for 2-5 days.

Period 2

Materials

3 of each of the medium listed in Period 1 per pair

Dropper bottles of 3% H2O2 and Kovac's reagent

One test bacterium from your instructor

Remember to observe all the test results for every culture examined, not just for the bacteria that you tested.

  1. Observe the fermentation broths. First, look for turbidity in the tube, record this as growth. Second, examine the color of the medium. The original color was purple (alkaline), the fermentation of the sugar to acid causes the medium to turn yellow (acidic). Record whether your organism ferments the sugar to acid. Finally look for a bubble in the Durham tube; if present it signifies the production of gas.
  2. Observe the streak on the starch agar plate. Then gently flood an area around growth with Gram's iodine. Allow the plate to react for about 2 min. and pour off the iodine solution into the sink. Starch will react with the iodine to form a blue complex. If the starch has been hydrolyzed by the extracellular enzyme amylase, a clear zone will be seen around the streak. Record this as a positive result. If the blue color runs all the way to the edge of the growth record this as a negative result.
  3. Observe the colonies on HIA and record your results. Are all the colonies of the same general type? If they are not, what does this mean? Describe the pigmentation, opacity, form, elevation and margin of the colonies. (Refer to Figure 7-4 for the correct terms to use.) After observing all nine cultures, perform the catalase test by adding one-half dropperful of 3% H2O2 to an area of the plate. Place the lid back on the plate and watch for the production of bubbles. The appearance of a constant evolution signifies the presence of the catalase enzyme.
  4. Carefully layer half a dropperful of Kovac's reagent onto the surface of the tryptone broth cultures and let sit for a few minutes. The development of a red ring at the broth reagent interface is a positive test for indole production. (Note that only one of the known cultures is expected to give a positive test for indole.)
  5. Nitrate reduction. To observe for the reduction of nitrate in the Nitrate Broth all the way to nitrogen gas (i.e., denitrification), look for a bubble within the Durham tube. To test for the reduction of nitrate only to nitrite, add a dropperful of each of the two nitrite reagents - sulfanilamide and N-(1-naphthyl)-ethylenediamine - to each of the tubes. Mix well. Any appearance of a red color (which may or may not persist) indicates the presence of nitrite. If no red color was seen, add a few more drops of each reagent, mix well, and observe again. If no indication of gas or nitrite was seen, the nitrate may not have been reduced, or it was reduced to a product for which we are not testing. Before discarding the tubes, one can check for unreduced nitrate by adding a small amount of granulated zinc to any tube not showing a positive reaction for nitrite or gas. Mix the tube well. If nitrate is present, it will be reduced by the zinc to nitrite which will react with the reagents already added, forming a pink or red color. (Disregard any gas generated with the addition of the zinc.) Do not record for the organism any reaction you see only upon addition of the zinc.
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