BIOLOGY Volume 2 - A Guide to General Biology - 2004

12. MICROBIOLOGY AND BIOTECHNOLOGY

12.9. Laboratory Work

12.9.2. Bacteriological Experiments

The three experiments described below, each utilizing aseptic techniques, are designed to help students master microbiological Methods. The first experiment—culturing lactic acid Bacteria—involves preparing Agar plates and streak plating. The second involves Gram staining of bacteria from Experiment 12.1 for examination under a Light Microscope. The third involves counting bacterial colonies using the serial dilution method.

Experiment 12.1. Examining the Bacterial Content of Fresh and Spoiled Milk

The purpose of this experiment is to determine how 24-hour storage of milk at room Temperature affects its quality and to discover why milk sours. Milk is practically a complete food for humans, and these experiments demonstrate that it also serves as an excellent nutrient medium for a wide range of bacteria.

Materials and Equipment

Four sterile Petri dishes containing nutrient agar

Inoculating loop

Bunsen burner

Permanent marker or wax pencil

Fresh pasteurized milk

Spoiled milk (milk left standing for 24 h at room temperature)

Incubator set to 35 °C

Procedure

1. Sterilize the loop in the Bunsen burner flame (hold it there until it glows red-hot) (Fig. 12.4).

2. Allow the loop to cool, then dip it into the fresh milk sample after thoroughly shaking it.

3. With your free hand, slightly lift the lid of the sterile agar plate and gently streak the Contents of the loop across the agar surface, as shown in Fig. 12.4.

4. Replace the lid and flame the loop again.

5. Label the bottom of the dish using a permanent marker (or wax pencil).

6. Repeat the entire procedure for a second plate using another sample of fresh milk.

7. Flame the loop again, let it cool, and dip it into the spoiled milk sample.

8. Streak the contents of the loop across The surface of a third plate and replace the lid.

9. Label the bottom of the dish with a permanent marker.

10. Repeat the same procedure with the fourth dish and the second sample of stale milk.

11. Place all dishes in an incubator and incubate at 35 °C for approximately three days. Before placing the dishes in the incubator, invert them to prevent Condensation droplets from falling onto the culture from above. After incubation, seal the two halves of each dish together with adhesive tape to prevent loss of the culture.

12. Describe the appearance of the colonies and compare your results with the description in Section 12.9.1.

Notes

1. Students may pour the plates themselves if sterile McCartney bottles containing molten agar are available.

2. When streaking, the number of bacteria in each subsequent streak gradually decreases. This method is more convenient to use when the bacterial count in the sample is high, such as in milk. It is typically employed for isolating pure bacterial colonies from a mixed culture.

3. After incubation, the dishes can be placed in a refrigerator and stored until needed. Refrigeration prevents further bacterial growth.

4. When the dishes are no longer needed, they should be placed in a disposal bag, autoclaved for 15 min, and only then discarded.

5. Other experiments can also be performed using milk. The experiment described above is the simplest one. The Effect of chilling can be studied. Furthermore, if samples of raw (unpasteurized) milk are readily obtainable (e.g., directly from a dairy farm), the effect of pasteurization on the bacterial content of milk can be investigated. For pasteurization, raw milk is poured into sterile test tubes, plugged with cotton wool, and then heated for 35 min at 63 °C in a Water bath. A third experimental variation is to incubate some of the dishes at 10 °C rather than 35 °C. Such a low temperature is favorable for the growth of Streptococcus lactis and does not support the growth of Lactobacillus.

Experiment 12.2. Staining bacteria for examination under a light microscope

Although live bacteria can be examined using a phase-contrast microscope, Cells are more commonly fixed and stained beforehand.

One staining method crucial for bacterial identification is the Gram stain. Before staining, bacteria are colorless. After staining, gram-positive bacteria appear purple, while gram-negative bacteria appear red. The difference between these Two Types of bacteria is described in Section 2.3.1 (Cell wall).

Materials and equipment

Primary stain — crystal violet (0.5% aqueous solution)

Mordant — Lugol's iodine solution

Acetone-alcohol (50:50 acetone:absolute alcohol) for decolorization

Counterstain — safranin (1% aqueous solution)

Inoculating loop, Bunsen burner

Thoroughly washed Glass slides (cleaned with alcohol)

Forceps

Staining rack placed over a sink or tray

Wash bottle with distilled water

Filter paper

Immersion oil and a microscope with an oil immersion objective

Procedure

(Steps 1–6 should take no more than 5 min) (From: Bacteriology, J. Humphries, John Murray, 1974.)

1. Prepare a bacterial smear on a glass microscope slide. To do this, flame a wire inoculation loop until red hot and let it cool. Place 1–2 drops of water in the center of a clean glass slide. Lightly Touch the loop to a bacterial colony of your choice from the previous experiment; open the Petri dish as little as possible to avoid contaminating the culture. Transfer the cells to the slide and mix gently with the loop. Spread the cells thinly across the slide to form a film covering an area of 3 x 1 cm. Re-sterilize the loop in the flame. Getting the smear thickness right is crucial: it should be barely opalescent and is much more likely to end up too thick than too thin. The thickness of the smear must be uniform across the entire surface. Allow the smear to air-dry completely (takes a few minutes).

2. Fix the bacteria. Hold the slide with forceps and, keeping it horizontal, pass it three times quickly through the yellow zone of a Bunsen burner flame. It is important not to overheat the slide. Check the temperature by tapping the slide gently against the back of your hand after each pass. If it feels warm rather than hot, the fixation is correct. Fixing kills the bacterial cells by coagulating their Cytoplasm and firmly adheres them to the slide.

3. Stain the bacteria. Staining can easily make a mess of the lab bench, so it is best performed on a staining rack placed over a sink or staining tray. You can improvise a rack by placing two glass or metal rods parallel to each other, exactly 5 cm apart, across the edges of the tray. Secure the rods with a little plasticine. Flood the slide with crystal violet solution and leave for 30 s. All bacteria will take up the stain and appear purple.

4. Wash off the excess stain with Lugol's iodine solution by flooding the slide and leaving it for 30 s. Rinse the iodine off with distilled water from a wash bottle. The iodine acts as a mordant, forming a complex with the dye that binds it more firmly inside the cells.

5. Decolorize the slide with an acetone-alcohol mixture until the dye stops washing out (approx. 3 s); immediately rinse with water afterward to prevent over-decolorizing the specimen. Repeat this step if necessary (mastering the exact decolorization time only comes with experience). Gram-negative bacteria lose their color during this step, while Gram-positive bacteria remain purple.

6. Flood the slide with safranin and leave for 1 min. Wash off the stain with water. Carefully blot the slide dry between sheets of clean filter paper and allow it to air-dry completely. Safranin acts as a counterstain. Applied after crystal violet, it stains all Gram-negative bacteria red.

7. Place a drop of immersion oil directly onto the smear and examine the specimen using an oil-immersion objective (section 5.11.2).

Results

Do your observations align with the information given in section 12.9.1 regarding bacterial counts in milk?

Experiment 12.3. Comparing bacterial numbers in fresh and spoiled milk

When a single bacterium is placed on nutrient agar, it begins to divide and eventually forms a colony that, unlike the original cell, is visible to the naked eye. This property can be utilized to enumerate bacteria.

Sterilize all equipment beforehand. The first part of the experiment relies on serial dilutions. Because the bacterial population in milk is massive, it is much more practical to take a small sample volume and dilute it by a known factor. Prepare a series of dilutions. In the second part of the experiment, take a sample from each dilution for plating. To determine the number of bacteria in a specific volume of milk (i.e., the titer), select the plate that shows an optimal number of colonies after incubation—high enough for statistical significance, but without overlapping.

Materials and Equipment

Six sterile Petri dishes with nutrient agar

Eight 1 cm³ graduated pipettes

One 10 cm³ graduated pipette

Six test tubes and a test tube rack

Cotton wool

Hot air oven set to 160 °C

Waterproof marker

Bunsen burner

100 cm³ of distilled water

Fresh milk

Spoiled milk

70% alcohol

Aluminum foil

Glass spreader

Equipment sterilization

1. Plug each of the six test tubes with a cotton stopper and wrap the stoppers tightly in aluminum foil.

2. Insert a small cotton plug into the tip of each of the eight 1 cm³ pipettes and one 10 cm³ pipette, then wrap each pipette individually in aluminum foil.

3. Place the stoppers and pipettes into a hot-air oven preheated to 160 °C and leave them there for 60 min (flasks containing media and water must not be sterilized in this type of oven).

4. Allow the equipment to cool before use.

Serial dilution of milk and plating onto agar plates

1. Label six sterile, stoppered test tubes as C1, C2, C3, H1, H2, and H3. Remove the aluminum foil from the stoppers.

2. Label the bottoms of six sterile nutrient agar plates as C1, C2, C3, H1, H2, and H3.

3. Add 9.9 cm³ of sterile distilled water to each of the six test tubes using the following procedure.

a) Remove the cotton stopper from the flask of sterile distilled water using the little and ring fingers of one hand.

b) Holding the stopper in one hand, take a sterile 10 cm³ pipette with the other hand and draw 9.9 cm³ of sterile distilled water from the flask.

c) Stopper the flask.

d) Remove the stopper from the first test tube using the exact same technique as described in step a.

e) Transfer 9.9 cm³ of water into this test tube.

f) Replace the stopper.

g) Repeat the procedure for the remaining five test tubes.

4. Shake the fresh milk sample thoroughly, take a sterile 1 cm³ pipette, and transfer 0.1 cm³ of milk into test tube C1. The stopper is removed and replaced in the same manner as before. This yields a 100-fold dilution.

5. Gently agitate the test tube to mix the contents thoroughly.

6. Take a new sterile pipette and transfer 0.1 cm³ from test tube C1 onto the sterile plate labeled C1, lifting the petri dish lid only slightly.

7. Sterilize the glass spreader by dipping it into 70% alcohol, allowing the excess alcohol to drain off, and then holding the spreader vertically in a Bunsen burner flame.

8. Allow the spreader to cool, and use the cooled spreader to spread the milk sample evenly across the surface of the agar plate.

9. Sterilize the spreader again.

10. Using the same pipette as in step 6, transfer 0,1 cm³ from test tube C1 to test tube C2, removing and replacing the cotton plug as described above.

11. Mix the sample thoroughly by shaking test tube C2. This yields a 10,000-fold dilution.

12. Repeat the operations described in steps 10–11, using C3 instead of C2. This gives a 1,000,000-fold dilution. Repeat the operations described in steps 6–9, using C3 instead of C1.

13. Repeat the serial dilutions using stale milk as the samples, and prepare agar plates H1, H2, and H3.

14. Invert the six prepared petri dishes and incubate them in an incubator at 35 °C for approximately 3 days.

15. Secure the lids and bases of the petri dishes with adhesive tape to prevent the potential spread of pathogens.

16. Assess bacterial growth on the plates. Wherever possible, count the number of individual colonies. Record your results in a table and use them to calculate the number of bacteria per 1 cm³ of undiluted milk.

Notes

See notes 3 and 4 at the end of the description for Experiment 12.1.