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Below is the protocol that we will follow for investigation of the Lactic Acid Bacteria.

Period 1

Materials

WORK IN PAIRS

Cultures of the following grown in APT Broth1 or Milk2:

Lactobacillus bulgaricus	Lactococcus lactis
Lactobacillus plantarum	Streptococcus thermophilus
Leuconostoc mesenteroides	Aerococcus viridans

Samples of juice (diluted 1/10) from 4 or 5-day-old and 10 to 12-day-old sauerkraut fermentations (Each pair uses either the younger or the older sample.)

2 tubes of APT Broth (approx. 8-10 ml)

2 tubes of pasteurized or sterilized milk (exactly 5 ml)

5 plates HIAG (HIA plus 5% glucose)

5 plates HIAS (HIA plus 5% sucrose, 0.5% glucose and 0.02% sodium azide)

4 dilution blanks (9 ml)

Pipettors and sterile tips

1 small, clean Erlenmeyer flask

Titration apparatus (with 0.1M NaOH and phenolphthalein)

1. Gram reaction and morphology. Prepare heat-fixed smears from the cultures. After performing the inoculations below, gram-stain the smears and determine the gram reaction, shape and arrangement of the cells for each culture. Reocrd your observations. A stained background will be seen for the organisms which had been grown in milk.
2. Differentiation between homo- and heterofermentative lactic acid bacteria. This simple test which detects CO₂ production by the heterofermentative lactics is helpful in the identifications of the genera. We will differentiate between the almost morphologically-identical genera Lactococcus and Leuconostoc.
3. Inoculate one tube of APT Broth with Lactococcus lactis and another tube with Leuconostoc mesenteroides.
4. Incubate the tubes in your desk drawer for 1-2 days. If the next lab period is more than 2 days away, place the tubes in the rack on the stage. (The tubes will be refrigerated until the day before the next lab period, then incubated at 30°C.)
5. Fermentation of milk. Some (not all!) lactic acid bacteria ferment lactose, the milk sugar.
6. The production of yogurt is accomplished by two organisms, Lactobacillus bulgaricus and Streptococcus thermophilus. The resulting low pH from lactose fermentation causes the coagula-tion of the milk protein (casein), forming a curd. The two organisms produce some flavoring compounds and also growth factors which assist their mutual growth. The following production of pseudo-yogurt will demonstrate the effect of these organisms on milk:
7. To each tube of milk (A and B), add 0.1 ml of L. bulgaricus and 0.1 ml of S. thermophilus. Incubate tube B at 37°C until the next period.
8. Add the contents of tube A to a clean Erlenmeyer flask. Obtain some distilled water in a clean glass from the special tap in the sink, and pipette 5 ml of the water into the flask. At the titration station, add 4 drops of phenolphthalein solution, mix well and titrate with 0.1M NaOH until the indicator shows a trace of pink color persisting for at least 15-20 seconds. Record the volume of NaOH used. Dispose the flask into the tray on the discard cart.
9. Slime production from sucrose. This procedure will demonstrate how some lactic acid bacteria produce slime from sucrose but not from most other sugars. In the case of Leuconostoc mesenteroides and certain other lactics, the enzyme dextran sucrase splits sucrose into glucose and fructose and also polymerizes the glucose into dextran.
10. Streak the culture of L. mesenteroides for isolated colonies on one plate each of glucose and sucrose-containing media, i.e., HIAG and HIAS. (See Materials. The sodium azide in the latter medium will be of no significance in this test.)
11. Incubate at 30°C until the next period.
12. Lactic acid bacteria in the sauerkraut fermentation. Sauerkraut is essentially shredded cabbage which has been altered by the growth and activities of bacteria indigenous to the cabbage (see the introduction). Important in the activities of these organisms and the reduction of spoilage organisms are anaerobic conditions and the initial addition of NaCl (2.5% of the total weight). Proceed as follows with one of the sauerkraut samples provided:
13. Prepare dilutions and platings of the sample (already a 1/10 dilution) onto HIAG and HIAS such that one plate of each medium is made for each of these plated dilutions: 10^-3, 10^-4, 10^-5 and 10^-6.
14. Optionally, make a streak plate of the sauerkraut sample, plating onto HIAS and HIAG.
15. Note that we are using Heart Infusion Agar plus glucose (HIAG) - an all-purpose medium - for the total count.
16. The other medium (HIAS) contains sodium azide and will support the growth of only lactic acid bacteria when incubated aerobically.
17. As HIAS contains sucrose, it will help to differentiate between the two major lactic acid genera in sauerkraut - the slime-forming Leuconostoc and the non-slime-forming Lactobacillus.
18. Incubate at 30°C until the next period.

Period 2

Materials

(per pair)

1 pipette (5 ml)

1 small, clean Erlenmeyer flask

titration apparatus (with 0.1M NaOH and phenolphthalein)

1. Differentiation between homo- and heterofermentative lactic acid bacteria. For each tube, plunge a red-hot inoculating loop fully into the culture. The evolution of gas indicates soluble CO₂ in the medium, a result of heterofermentative metabolism. This test will differentiate effectively between Leuconostoc and the other chain-forming genera of cocci in the lactic acid bacteria group. Observe Figure 12-2 to see a movie of the hot loop test. Record your results (Additional note: Pediococcus and Aerococcus are both homofermentative; Lactobacillus species are either homo- or heterofermentative.)
2. Analysis of the milk fermentation. Note the odor and texture of the pseudo-yogurt in tube B incubated since last period. Titrate as you did for tube A; the 5 ml of distilled water can be added to the tube to help loosen the solid curd.
3. Compare the results of both tubes. Has acid been produced? What effect has this had on the milk?
4. From the amount of NaOH used, the percent lactic acid in the milk can be calculated according to the following formula: (Recall that the sample volume was 5 ml.)

Lactic Acid Formula:

5. Slime production from sucrose. Describe the colonial morphology of Leuconostoc mesenteroides on each medium (glucose vs. sucrose). Poke at the growth on each plate with a sterile loop and note the relative consistencies of the growth. Record and explain your observations.
6. Lactic acid bacteria in the sauerkraut fermentation. If the colonies are difficult to see, reincubate the plates at room temperature and make your observations next period.
7. From the colony counts on HIAG and HIAS, determine the CFUs per ml in the original, undiluted sample for the total count and the count of lactic acid bacteria.
8. Optionally, observe your streak plates of the sauerkraut on the streak plates of HIAS or HIAG.
9. Are any relatively slimy or mucoid colonies apparent on the medium containing sucrose (HIAS)? If so, determine the CFUs per ml of Leuconostoc in the sample.
10. Compare your results with those who used the other sauerkraut sample. What may the relative differences in the various counts mean?

An example of a hot loop test

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